Anti-trem2 antibodies and methods of use thereof

ABSTRACT

In one aspect, antibodies that specifically bind to a human triggering receptor expressed on myeloid cells 2 (TREM2) protein are provided. In some embodiments, the antibody decreases levels of soluble TREM2 (sTREM2). In some embodiments, the antibody enhances TREM2 activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US2020/019093, filed Feb. 20, 2020, which claims priority to U.S.Provisional Application No. 62/808,141, filed Feb. 20, 2019, thedisclosures of which are hereby incorporated by reference in theirentirety for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 10, 2021, isnamed 102342-003310US-1262289 SL.txt and is 55,956 bytes in size.

BACKGROUND

Triggering receptor expressed on myeloid cells-2 (TREM2) is atransmembrane receptor that is expressed on microglia and is believed tofunction in regulating phagocytosis, cell survival, and the productionof pro-inflammatory cytokines. Mutations in TREM2 have been identifiedin neurodegenerative diseases including Alzheimer's disease, Nasu-Hakoladisease, Parkinson's disease, amyotrophic lateral sclerosis, andfrontotemporal dementia. Additionally, altered levels of soluble TREM2(sTREM2) have been reported in the cerebrospinal fluid of patientshaving Alzheimer's disease or frontotemporal dementia who have amutation in TREM2.

There remains a need for therapeutic agents that modulate TREM2 activityor levels of sTREM2.

BRIEF SUMMARY

In one aspect, isolated antibodies or antigen-binding fragments thereofthat specifically binds to a human triggering receptor expressed onmyeloid cells 2 (TREM2) are provided. In some embodiments, the antibodyor antigen-binding fragment thereof that specifically binds to TREM2comprises:

-   -   (a) a CDR-H1 sequence comprising the sequence of GFSIEDFYIH (SEQ        ID NO:29);    -   (b) a CDR-H2 sequence comprising the sequence of        W-I-D-P-E-β₆-G-β₈-S-K-Y-A-P-K-F-Q-G (SEQ ID NO:47), wherein β₆        is N or Q and β₈ is D or E;    -   (c) a CDR-H3 sequence comprising the sequence of HADHGNYGSTMDY        (SEQ ID NO:31);    -   (d) a CDR-L1 sequence comprising the sequence of HASQHINVWLS        (SEQ ID NO:32);    -   (e) a CDR-L2 sequence comprising the sequence of KASNLHT (SEQ ID        NO:33); and    -   (f) a CDR-L3 sequence comprising the sequence of QQGQTYPRT (SEQ        ID NO:34).

In some embodiments, the CDR-H2 sequence is selected from SEQ ID NOS:30,39, 41, and 43.

In some embodiments, the antibody or antigen-binding fragment comprises:

-   -   (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:29,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:30, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:31, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:32, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:33, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:34; or    -   (b) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:29,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:39, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:31, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:32, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:33, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:34; or    -   (c) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:29,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:41, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:31, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:32, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:33, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:34; or    -   (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:29,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:43, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:31, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:32, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:33, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:34.

In some embodiments, the antibody or antigen-binding fragment comprisesa V_(H) sequence that has at least 85% sequence identity to any one ofSEQ ID NOS:27, 35, 37, 38, 40, 42, 44, 45, and 46. In some embodiments,the V_(H) sequence has at least 90% sequence identity to SEQ ID NO:27.In some embodiments, the V_(H) sequence has at least 95% sequenceidentity to SEQ ID NO:27. In some embodiments, the V_(H) sequencecomprises SEQ ID NO:27. In some embodiments, the V_(H) sequence has atleast 90% sequence identity to SEQ ID NO:42. In some embodiments, theV_(H) sequence has at least 95% sequence identity to SEQ ID NO:42. Insome embodiments, the V_(H) sequence comprises SEQ ID NO:42. In someembodiments, the V_(H) sequence has at least 90% sequence identity toSEQ ID NO:45. In some embodiments, the V_(H) sequence has at least 95%sequence identity to SEQ ID NO:45. In some embodiments, the V_(H)sequence comprises SEQ ID NO:45.

In some embodiments, the antibody or antigen-binding fragment comprisesa V_(L) sequence that has at least 85% sequence identity to SEQ ID NO:28or SEQ ID NO:36. In some embodiments, the V_(L) sequence has at least90% sequence identity to SEQ ID NO:28. In some embodiments, the V_(L)sequence has at least 95% sequence identity to SEQ ID NO:28. In someembodiments, the V_(L) sequence comprises SEQ ID NO:28. In someembodiments, the V_(L) sequence has at least 90% sequence identity toSEQ ID NO:36. In some embodiments, the V_(L) sequence has at least 95%sequence identity to SEQ ID NO:36. In some embodiments, the V_(L)sequence comprises SEQ ID NO:36.

In some embodiments, the antibody or antigen-binding fragment comprises:

-   -   (a) a V_(H) sequence comprising SEQ ID NO:27 and a V_(L)        sequence comprising SEQ ID NO:28; or    -   (b) a V_(H) sequence comprising SEQ ID NO:35 and a V_(L)        sequence comprising SEQ ID NO:36; or    -   (c) a V_(H) sequence comprising SEQ ID NO:37 and a V_(L)        sequence comprising SEQ ID NO:36; or    -   (d) a V_(H) sequence comprising SEQ ID NO:38 and a V_(L)        sequence comprising SEQ ID NO:36; or    -   (e) a V_(H) sequence comprising SEQ ID NO:40 and a V_(L)        sequence comprising SEQ ID NO:36; or    -   (f) a V_(H) sequence comprising SEQ ID NO:42 and a V_(L)        sequence comprising SEQ ID NO:36; or    -   (g) a V_(H) sequence comprising SEQ ID NO:44 and a V_(L)        sequence comprising SEQ ID NO:36; or    -   (h) a V_(H) sequence comprising SEQ ID NO:45 and a V_(L)        sequence comprising SEQ ID NO:36; or    -   (i) a V_(H) sequence comprising SEQ ID NO:46 and a V_(L)        sequence comprising SEQ ID NO:36.

In some embodiments, an antibody or antigen-binding fragment thereofthat specifically binds to TREM2 comprises:

-   -   (a) a CDR-H1 sequence comprising the sequence of        G-F-T-F-T-α₆-F-Y-M-S(SEQ ID NO:48), wherein α₆ is D or N;    -   (b) a CDR-H2 sequence comprising the sequence of        V—I-R-N-β₅-β₆-N-β₈-Y-Tβ₁₁-β₁₂-Y—N-P-S—V-K-G (SEQ ID NO:49),        wherein β₅ is K or R; β₆ is A or P; β₈ is G or A; β₁₁ is A or T;        and β₁₂ is G or D;    -   (c) a CDR-H3 sequence comprising the sequence of        γ₁-R-L-γ₄-Y-G-F-D-Y (SEQ ID NO:50), wherein γ₁ is A or T; and γ₄        is T or S;    -   (d) a CDR-L1 sequence comprising the sequence of        Q-S—S-K-S-L-L-H-S-δ₁₀-G-K-T-Y-L-N(SEQ ID NO:51), wherein δ₁₀ is        N or T;    -   (e) a CDR-L2 sequence comprising the sequence of WMSTRAS (SEQ ID        NO:8); and    -   (f) a CDR-L3 sequence comprising the sequence of        Q-Q-F-L-E-ϕ₆-P-F-T (SEQ ID NO:52), wherein ϕ₆ is Y or F.

In some embodiments, the CDR-H1 sequence is selected from any one of SEQID NOS:4 and 12. In some embodiments, the CDR-H2 sequence is selectedfrom any one of SEQ ID NOS:5, 13, and 25. In some embodiments, theCDR-H3 sequence is selected from any one of SEQ ID NOS:6, 14, and 17. Insome embodiments, the CDR-L1 sequence is selected from any one of SEQ IDNOS:7 and 23. In some embodiments, the CDR-L3 sequence is selected fromany one of SEQ ID NOS:9 and 18.

In some embodiments, the antibody or antigen-binding fragment comprises:

-   -   (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:5, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:17, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:18; or    -   (b) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:5, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:17, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:23, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:18; or    -   (c) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:25, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:17, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:18; or    -   (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:25, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:17, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:23, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:18; or    -   (e) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:5, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:6, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:9; or    -   (f) a a CDR-H1 comprising the amino acid sequence of SEQ ID        NO:12, a CDR-H2 comprising the amino acid sequence of SEQ ID        NO:13, a CDR-H3 comprising the amino acid sequence of SEQ ID        NO:14, a CDR-L1 comprising the amino acid sequence of SEQ ID        NO:7, a CDR-L2 comprising the amino acid sequence of SEQ ID        NO:8, and a CDR-L3 comprising the amino acid sequence of SEQ ID        NO:9; or    -   (g) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:25, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:17, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:9.

In some embodiments, the antibody or antigen-binding fragment comprisesa V_(H) sequence that has at least 85% sequence identity to any one ofSEQ ID NOS:2, 10, 15, 19, 21, 24, 26, and 79. In some embodiments, theV_(H) sequence has at least 90% sequence identity to SEQ ID NO:15. Insome embodiments, the V_(H) sequence has at least 95% sequence identityto SEQ ID NO:15. In some embodiments, the V_(H) sequence comprises SEQID NO:15. In some embodiments, the V_(H) sequence has at least 90%sequence identity to SEQ ID NO:24. In some embodiments, the V_(H)sequence has at least 95% sequence identity to SEQ ID NO:24. In someembodiments, the V_(H) sequence comprises SEQ ID NO:24. In someembodiments, the V_(H) sequence has at least 90% sequence identity toSEQ ID NO:79. In some embodiments, the V_(H) sequence has at least 95%sequence identity to SEQ ID NO:79. In some embodiments, the V_(H)sequence comprises SEQ ID NO:79.

In some embodiments, the antibody or antigen-binding fragment comprisesa V_(L) sequence that has at least 85% sequence identity to any one ofSEQ ID NOS:3, 11, 16, 20, 22, and 68. In some embodiments, the V_(L)sequence has at least 90% sequence identity to SEQ ID NO:16. In someembodiments, the V_(L) sequence has at least 95% sequence identity toSEQ ID NO:16. In some embodiments, the V_(L) sequence comprises SEQ IDNO:16. In some embodiments, the V_(L) sequence has at least 90% sequenceidentity to SEQ ID NO:22. In some embodiments, the V_(L) sequence has atleast 95% sequence identity to SEQ ID NO:22. In some embodiments, theV_(L) sequence comprises SEQ ID NO:22. In some embodiments, the V_(L)sequence has at least 90% sequence identity to SEQ ID NO:68. In someembodiments, the V_(L) sequence has at least 95% sequence identity toSEQ ID NO:68. In some embodiments, the V_(L) sequence comprises SEQ IDNO:68.

In some embodiments, the antibody or antigen-binding fragment comprises:

-   -   (a) a V_(H) sequence comprising SEQ ID NO:15 and a V_(L)        sequence comprising SEQ ID NO:16; or    -   (b) a V_(H) sequence comprising SEQ ID NO:19 and a V_(L)        sequence comprising SEQ ID NO:20; or    -   (c) a V_(H) sequence comprising SEQ ID NO:21 and a V_(L)        sequence comprising SEQ ID NO:20; or    -   (d) a V_(H) sequence comprising SEQ ID NO:19 and a V_(L)        sequence comprising SEQ ID NO:22; or    -   (e) a V_(H) sequence comprising SEQ ID NO:79 and a V_(L)        sequence comprising SEQ ID NO:22; or    -   (f) a V_(H) sequence comprising SEQ ID NO:24 and a V_(L)        sequence comprising SEQ ID NO:20; or    -   (g) a V_(H) sequence comprising SEQ ID NO:26 and a V_(L)        sequence comprising SEQ ID NO:20; or    -   (h) a V_(H) sequence comprising SEQ ID NO:24 and a V_(L)        sequence comprising SEQ ID NO:22; or    -   (i) a V_(H) sequence comprising SEQ ID NO:26 and a V_(L)        sequence comprising SEQ ID NO:22; or    -   (j) a V_(H) sequence comprising SEQ ID NO:2 and a V_(L) sequence        comprising SEQ ID NO:3; or    -   (k) a V_(H) sequence comprising SEQ ID NO:10 and a V_(L)        sequence comprising SEQ ID NO:11; or    -   (l) a V_(H) sequence comprising SEQ ID NO:24 and a V_(L)        sequence comprising SEQ ID NO:68.

In some embodiments, an antibody or antigen-binding fragment thereofthat specifically binds to TREM2 comprises:

-   -   (a) a CDR-H1 sequence comprising the amino acid sequence of any        one of SEQ ID NOS:4, 12, and 29;    -   (b) a CDR-H2 sequence comprising the amino acid sequence of any        one of SEQ ID NOS:5, 13, 25, 30, 39, 41, and 43;    -   (c) a CDR-H3 sequence comprising the amino acid sequence of any        one of SEQ ID NOS:6, 14, 17, and 31;    -   (d) a CDR-L1 sequence comprising the amino acid sequence of any        one of SEQ ID NOS:7, 23, and 32;    -   (e) a CDR-L2 sequence comprising the amino acid sequence of any        one of SEQ ID NOS:8 and 33; and    -   (f) a CDR-L3 sequence comprising the amino acid sequence of any        one of SEQ ID NOS:9, 18, and 34.

In some embodiments, the antibody or antigen-binding fragment comprises:

-   -   (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:5, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:6, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:9; or    -   (b) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:5, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:17, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:18; or    -   (c) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:5, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:17, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:23, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:18; or    -   (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:25, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:17, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:18; or    -   (e) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:25, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:17, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:23, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:18; or    -   (f) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:12,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:13, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:14, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:9;    -   (g) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:29,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:30, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:31, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:32, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:33, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:34; or    -   (h) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:29,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:39, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:31, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:32, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:33, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:34; or    -   (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:29,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:41, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:31, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:32, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:33, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:34; or    -   (j) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:29,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:43, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:31, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:32, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:33, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:34; or    -   (k) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:25, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:17, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:9.

In some embodiments, the antibody or antigen-binding fragment comprisesa heavy chain variable region that has at least 85% sequence identity toany one of SEQ ID NOS:2, 10, 15, 19, 21, 24, 26, 27, 35, 37, 38, 40, 42,44, 45, 46, and 79. In some embodiments, the antibody or antigen-bindingfragment comprises a light chain variable region that has at least 85%sequence identity to any one of SEQ ID NOS:3, 11, 16, 20, 22, 28, 36,and 68.

In some embodiments, the antibody or antigen-binding fragment comprises:

-   -   (a) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:2 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:3; or    -   (b) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:10 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:11; or    -   (c) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:15 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:16; or    -   (d) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:19 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:20; or    -   (e) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:21 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:20; or    -   (f) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:19 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:22; or    -   (g) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:79 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:22; or    -   (h) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:24 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:20; or    -   (i) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:26 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:20; or    -   (j) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:24 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:22; or    -   (k) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:26 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:22; or    -   (l) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:27 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:28; or    -   (m) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:35 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:36; or    -   (n) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:37 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:36; or    -   (o) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:38 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:36; or    -   (p) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:40 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:36; or    -   (q) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:42 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:36; or    -   (r) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:44 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:36; or    -   (s) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:45 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:36; or    -   (t) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:46 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:36; or    -   (u) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:24 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:68.

In some embodiments, an antibody or antigen-binding fragment thereofthat specifically binds to TREM2 recognizes an epitope that is the sameor substantially the same as the epitope recognized by antibody cloneselected from the group consisting of: Clone CL0020306, Clone CL0020188,Clone CL0020188-1, Clone CL0020188-2, Clone CL0020188-3, CloneCL0020188-4, Clone CL0020188-5, Clone CL0020188-6, Clone CL0020188-7,Clone CL0020188-8, Clone CL0020307, Clone CL0020123, Clone CL0020123-1,Clone CL0020123-2, Clone CL0020123-3, Clone CL0020123-4, CloneCL0020123-5, Clone CL0020123-6, Clone CL0020123-7, and CloneCL0020123-8.

In some embodiments, the antibody or antigen-binding fragment recognizesan epitope that is the same or substantially the same as the epitoperecognized by an antibody clone selected from the group consisting of:Clone CL0020123, Clone CL0020123-1, Clone CL0020123-2, CloneCL0020123-3, Clone CL0020123-4, Clone CL0020123-5, Clone CL0020123-6,Clone CL0020123-7, and Clone CL0020123-8. In particular embodiments, theantibody or antigen-binding fragment recognizes one or more of thefollowing epitopes in SEQ ID NO:1: (i) amino acid residues 55-63(GEKGPCQRV (SEQ ID NO:70)), (ii) amino acids 96-107 (TLRNLQPHDAGL (SEQID NO:71)), and (iii) amino acid residues 126-129 (VEVL (SEQ ID NO:72)).In another aspect, the disclosure features an isolated antibody orantigen-binding fragment thereof that specifically binds to a humanTREM2, wherein the antibody or antigen-binding fragment thereofrecognizes an epitope comprising or consisting of one or more of thefollowing epitopes in SEQ ID NO:1: (i) amino acid residues 55-63(GEKGPCQRV (SEQ ID NO:70)), (ii) amino acids 96-107 (TLRNLQPHDAGL (SEQID NO:71)), and (iii) amino acid residues 126-129 (VEVL (SEQ ID NO:72)).In some embodiments, the antibody or antigen-binding fragment recognizesan epitope that is the same or substantially the same as the epitoperecognized by an antibody clone selected from the group consisting of:Clone CL0020188, Clone CL0020188-1, Clone CL0020188-2, CloneCL0020188-3, Clone CL0020188-4, Clone CL0020188-5, Clone CL0020188-6,Clone CL0020188-7, Clone CL0020188-8, Clone CL0020307, and CloneCL0020306. In particular embodiments, the antibody or antigen-bindingfragment recognizes amino acid residues 143-149 (FPGESES (SEQ ID NO:69))in SEQ ID NO:1. In another aspect, the disclosure features an isolatedantibody or antigen-binding fragment thereof that specifically binds toa human TREM2, wherein the antibody or antigen-binding fragment thereofrecognizes an epitope comprising or consisting of amino acid residues143-149 (FPGESES (SEQ ID NO:69)) in SEQ ID NO:1.

In some embodiments, an antibody or antigen-binding fragment asdisclosed herein decreases levels of soluble TREM2 protein (sTREM2). Insome embodiments, an antibody or antigen-binding fragment as disclosedherein binds soluble TREM2 protein (sTREM2) in healthy human CSF orcynomolgus CSF with better potency compared to a reference antibody. Insome embodiments, the reference antibody is represented by a combinationof sequences selected from the group consisting of: SEQ ID NOS:73 and74; SEQ ID NOS:75 and 76; and SEQ ID NOS:77 and 78. In some embodiments,the potency assay is carried out substantially as described in Example11. In some embodiments, an antibody or antigen-binding fragment asdisclosed herein enhances TREM2 activity. In some embodiments, theantibody or antigen-binding fragment thereof enhances phagocytosis orenhances the migration, differentiation, function, or survival ofmyeloid cells, microglia, or macrophages. In some embodiments, theantibody or antigen-binding fragment thereof enhances microglia functionwithout increasing neuroinflammation. In some embodiments, the antibodyor antigen-binding fragment thereof enhances Syk phosphorylation. Insome embodiments, the antibody or antigen-binding fragment thereofenhances Syk phosphorylation in the presence of a TREM2 ligand. In someembodiments, the antibody or antigen-binding fragment thereof exhibitscross-reactivity with a cynomolgus TREM2 protein.

In some embodiments, an antibody or antigen-binding fragment asdisclosed herein is a monoclonal antibody. In some embodiments, anantibody or antigen-binding fragment as disclosed herein is a chimericantibody. In some embodiments, an antibody or antigen-binding fragmentas disclosed herein is a humanized antibody. In some embodiments, anantibody or antigen-binding fragment as disclosed herein is a fullyhuman antibody. In some embodiments, an antibody or antigen-bindingfragment as disclosed herein is a Fab, a F(ab′)2, a scFv, or a bivalentscFv.

In another aspect, the disclosure provides antibodies or antigen-bindingfragments thereof that competes with an isolated anti-TREM2 antibody asdisclosed herein for binding to the human TREM2 protein.

In another aspect, the disclosure provides pharmaceutical compositionscomprising an antibody or antigen-binding fragment as disclosed hereinthat specifically binds to TREM2 and a pharmaceutically acceptablecarrier.

In yet another aspect, the disclosure provides kits comprising: anantibody or antigen-binding fragment as disclosed herein thatspecifically binds to TREM2 or a pharmaceutical composition comprisingthe anti-TREM2 antibody or antigen-binding fragment; and instructionsfor use thereof.

In still another aspect, the disclosure provides methods of treating aneurodegenerative disease in a subject. In some embodiments, the methodcomprises administering to the subject an anti-TREM2 antibody orantigen-binding fragment as disclosed herein or a pharmaceuticalcomposition comprising an anti-TREM2 antibody or antigen-bindingfragment as disclosed herein.

In some embodiments, the neurodegenerative disease is selected from thegroup consisting of: Alzheimer's disease, primary age-related tauopathy,progressive supranuclear palsy (PSP), frontotemporal dementia,frontotemporal dementia with parkinsonism linked to chromosome 17,argyrophilic grain dementia, amyotrophic lateral sclerosis, amyotrophiclateral sclerosis/parkinsonism-dementia complex of Guam (ALS-PDC),corticobasal degeneration, chronic traumatic encephalopathy,Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillarytangles with calcification, Down's syndrome, familial British dementia,familial Danish dementia, Gerstmann-Straussler-Scheinker disease,globular glial tauopathy, Guadeloupean parkinsonism with dementia,Guadelopean PSP, Hallevorden-Spatz disease, hereditary diffuseleukoencephalopathy with spheroids (HDLS), Huntington's disease,inclusion-body myositis, multiple system atrophy, myotonic dystrophy,Nasu-Hakola disease, neurofibrillary tangle-predominant dementia,Niemann-Pick disease type C, pallido-ponto-nigral degeneration,Parkinson's disease, Pick's disease, postencephalitic parkinsonism,prion protein cerebral amyloid angiopathy, progressive subcorticalgliosis, subacute sclerosing panencephalitis, and tangle only dementia.

In yet another aspect, the disclosure provides methods of decreasinglevels of sTREM2 in a subject having a neurodegenerative disease. Insome embodiments, the method comprises administering to the subject ananti-TREM2 antibody or antigen-binding fragment as disclosed herein or apharmaceutical composition comprising an anti-TREM2 antibody orantigen-binding fragment as disclosed herein.

In still another aspect, the disclosure provides methods of enhancingTREM2 activity in a subject having a neurodegenerative disease. In someembodiments, the method comprises administering to the subject ananti-TREM2 antibody or antigen-binding fragment as disclosed herein or apharmaceutical composition comprising an anti-TREM2 antibody orantigen-binding fragment as disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 includes representative flow cytometry histograms representingbinding of an exemplary anti-TREM2 antibody to surface TREM2 on HEKcells expressing TREM2.

FIG. 2 includes a representative dose-response curve of pSyk signalactivation by an exemplary anti-TREM2 antibody in primary humanmacrophage cells. Solid black circles (•) represent anti-TREM2 antibody,and open white circles)(° represent isotype control.

FIGS. 3A and 3B include representative dose-response curves of pSyksignal activation in human iPSC microglia cells after pre-treatment withexemplary anti-TREM2 antibodies for 5 minutes (FIG. 3A) or for 24 hours(FIG. 3B), followed by dosing with lipid vesicles and assessment ofliposome response in the cells.

FIG. 4 includes representative dose-response curves of NFAT-luciferasereporter activity in Jurkat NFAT cells expressing human TREM2/DAP12 inresponse to stimulation by exemplary anti-TREM2 antibodies. Solid blackcircles (•) represent anti-TREM2 antibody, and open white circles)(°represent isotype control.

FIG. 5 illustrates representative dose-response curves of cell survivalin human macrophage cells in response to treatment with exemplaryanti-TREM2 antibodies.

FIG. 6 illustrates representative soluble TREM2 levels (sTREM2) as afunction of the anti-TREM2 antibody concentration for exemplaryanti-TREM2 antibodies.

FIG. 7 is a bar chart indicating mean pHrodo fluorescence intensity percell in human macrophages treated with exemplary anti-TREM2 antibodies.

FIG. 8A is a representative microscopy image of lipid accumulation iniPSC microglia treated with myelin, followed by incubation withexemplary anti-TREM2 antibody or isotype control.

FIG. 8B is a representative bar chart of Nile Red staining (indicatinglipid accumulation) of iPSC microglia that were imaged in FIG. 8A.

FIGS. 8C-8F include bar charts illustrating quantified levels ofcholesteryl ester species (FIGS. 8C and 8E) and triacylglyceride lipidspecies (FIGS. 8D and 8F) in iPSC microglia treated with myelin,followed by incubation with exemplary anti-TREM2 antibodies. FIGS. 8Eand 8F represent data for iPSC microglia for which a myelin washout stepwas included prior to incubation with the exemplary anti-TREM2antibodies.

FIG. 9 includes representative mouse plasma pharmacokinetic profiles ofexemplary anti-TREM2 antibodies.

FIGS. 10A and 10B include bar charts illustrating change in totalsoluble TREM2 (sTREM2) (FIG. 10A) and antibody-bound TREM2 (FIG. 10B) inmouse plasma for exemplary anti-TREM2 antibodies, which were injectedinto TREM2 cDNA KI (huTrem2^(KI/KI)) mice.

FIGS. 11A and 11B include dose-response binding curves to human TREM2 inHEK cells for exemplary humanized and sequence-optimized anti-TREM2antibodies.

FIGS. 12A and 12B include dose-response curves of pSyk signal activationby exemplary humanized and sequence-optimized anti-TREM2 antibodies inHEK293-H6 cells.

FIG. 13 illustrates dose-response curves of cell survival in humanmacrophage cells in response to treatment with exemplary humanized andsequence-optimized anti-TREM2 antibodies.

FIGS. 14A and 14B include dose-response curves of lipid clearance iniPSC microglia in response to treatment with exemplary humanized andsequence-optimized anti-TREM2 antibodies.

DETAILED DESCRIPTION I. Introduction

TREM2 is a transmembrane receptor that is expressed on the cell surfaceof microglia, dendritic cells, macrophages, and osteoclasts. Withoutbeing bound to a particular theory, it is believed that upon ligandbinding, TREM2 forms a signaling complex with a transmembrane adapterprotein, DNAX-activating protein 12 (DAP12), which in turn is tyrosinephosphorylated by the protein kinase SRC. It is believed that theactivated TREM2/DAP12 signaling complex mediates intracellular signalingby recruiting and phosphorylating kinases such as Syk kinase.TREM2/DAP12 signaling modulates activities such as phagocytosis, cellgrowth and survival, pro-inflammatory cytokine secretion, and themigration of cells such as microglia and macrophages. TREM2 undergoesregulated intramembrane proteolysis, in which the membrane-associatedfull-length TREM2 is cleaved by the metalloprotease ADAM10 into a sTREM2portion that is shed from the cell and a membrane-retained C-terminalfragment that is further degraded by a gamma-secretase. Altered levelsof sTREM2 have been reported in patients having Alzheimer's disease orfrontotemporal dementia and having a mutation in TREM2. Additionally,mutations in TREM2 are associated with altered functions such asimpaired phagocytosis and reduced microglial function.

As detailed in the Examples section below, antibodies have beengenerated that specifically bind to human TREM2 and that modulate one ormore downstream functions of the TREM2/DAP12 signaling complex.Accordingly, in one aspect, the present disclosure provides anti-TREM2antibodies and antigen-binding fragments thereof. Accordingly, in oneaspect, the present disclosure provides anti-TREM2 antibodies andantigen-binding portions thereof.

In some embodiments, the anti-TREM2 antibodies enhance TREM2 activity,e.g., enhance phagocytosis or enhance the differentiation, function,migration, or survival of myeloid cells, microglia, or macrophages.Thus, in another aspect, methods of enhancing TREM2 activity, e.g., in asubject having a neurodegenerative disease, are provided.

In some embodiments, the anti-TREM2 antibodies reduce shedding ofsTREM2. Thus, in another aspect, methods of decreasing levels of sTREM2,e.g., in a subject having a neurodegenerative disease, are provided.

II. Definitions

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the content clearly dictates otherwise. Thus, forexample, reference to “an antibody” optionally includes a combination oftwo or more such molecules, and the like.

As used herein, the terms “about” and “approximately,” when used tomodify an amount specified in a numeric value or range, indicate thatthe numeric value as well as reasonable deviations from the value knownto the skilled person in the art, for example ±20%, ±10%, or ±5%, arewithin the intended meaning of the recited value.

As used herein, the term “TREM2 protein” refers to a triggering receptorexpressed on myeloid cells 2 protein that is encoded by the gene TREM2.As used herein, a “TREM2 protein” refers to a native (i.e., wild-type)TREM2 protein of any vertebrate, such as but not limited to human,non-human primates (e.g., cynomolgus monkey), rodents (e.g., mice, rat),and other mammals. In some embodiments, a TREM2 protein is a human TREM2protein having the sequence identified in UniprotKB accession numberQ9NZC2 (SEQ ID NO:1).

As used herein, the term “anti-TREM2 antibody” refers to an antibodythat specifically binds to a TREM2 protein (e.g., human TREM2).

As used herein, the term “antibody” refers to a protein with animmunoglobulin fold that specifically binds to an antigen via itsvariable regions. The term encompasses intact polyclonal antibodies,intact monoclonal antibodies, single chain antibodies, multispecificantibodies such as bispecific antibodies, monospecific antibodies,monovalent antibodies, chimeric antibodies, humanized antibodies, andhuman antibodies. The term “antibody,” as used herein, also includesantibody fragments that retain binding specificity via its variableregions, including but not limited to Fab, F(ab′)₂, Fv, scFv, andbivalent scFv. Antibodies can contain light chains that are classifiedas either kappa or lambda. Antibodies can contain heavy chains that areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms “variable light chain”(VL) and “variable heavy chain” (V_(H)) refer to these light and heavychains, respectively.

The term “variable region” or “variable domain” refers to a domain in anantibody heavy chain or light chain that is derived from a germlineVariable (V) gene, Diversity (D) gene, or Joining (J) gene (and notderived from a Constant (Cμ and Cδ) gene segment), and that gives anantibody its specificity for binding to an antigen. Typically, anantibody variable region comprises four conserved “framework” regionsinterspersed with three hypervariable “complementarity determiningregions.”

The term “complementarity determining region” or “CDR” refers to thethree hypervariable regions in each chain that interrupt the fourframework regions established by the light and heavy chain variableregions. The CDRs are primarily responsible for antibody binding to anepitope of an antigen. The CDRs of each chain are typically referred toas CDR1, CDR2, and CDR3, numbered sequentially starting from theN-terminus, and are also typically identified by the chain in which theparticular CDR is located. Thus, a V_(H) CDR3 or CDR-H3 is located inthe variable region of the heavy chain of the antibody in which it isfound, whereas a V_(L) CDR1 or CDR-L1 is the CDR1 from the variableregion of the light chain of the antibody in which it is found.

The “framework regions” or “FRs” of different light or heavy chains arerelatively conserved within a species. The framework region of anantibody, that is the combined framework regions of the constituentlight and heavy chains, serves to position and align the CDRs inthree-dimensional space. Framework sequences can be obtained from publicDNA databases or published references that include germline antibodygene sequences. For example, germline DNA sequences for human heavy andlight chain variable region genes can be found in the “VBASE2” germlinevariable gene sequence database for human and mouse sequences.

The amino acid sequences of the CDRs and framework regions can bedetermined using various well-known definitions in the art, e.g., Kabat,Chothia, international ImMunoGeneTics database (IMGT), AbM, and observedantigen contacts (“Contact”). In some embodiments, CDRs are determinedaccording to the Contact definition. See, MacCallum et al., J. Mol.Biol., 262:732-745 (1996). In some embodiments, CDRs are determined by acombination of Kabat, Chothia, and/or Contact CDR definitions.

The terms “antigen-binding portion” and “antigen-binding fragment” areused interchangeably herein and refer to one or more fragments of anantibody that retains the ability to specifically bind to an antigen(e.g., a TREM2 protein) via its variable region. Examples ofantigen-binding fragments include, but are not limited to, a Fabfragment (a monovalent fragment consisting of the VL, V_(H), CL and CH1domains), F(ab′)2 fragment (a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region), singlechain Fv (scFv), disulfide-linked Fv (dsFv), complementarity determiningregions (CDRs), a V_(L) (light chain variable region), and a V_(H)(heavy chain variable region).

The term “epitope” refers to the area or region of an antigen to whichthe CDRs of an antibody specifically binds and can include a few aminoacids or portions of a few amino acids, e.g., 5 or 6, or more, e.g., 20or more amino acids, or portions of those amino acids. For example,where the target is a protein, the epitope can be comprised ofconsecutive amino acids (e.g., a linear epitope), or amino acids fromdifferent parts of the protein that are brought into proximity byprotein folding (e.g., a discontinuous or conformational epitope). Insome embodiments, the epitope is phosphorylated at one amino acid (e.g.,at a serine or threonine residue).

As used herein, the phrase “recognizes an epitope,” as used withreference to an anti-TREM2 antibody, means that the antibody CDRsinteract with or specifically bind to the antigen (i.e., the TREM2protein) at that epitope or a portion of the antigen containing thatepitope.

As used herein, the term “multispecific antibody” refers to an antibodythat comprises two or more different antigen-binding portions, in whicheach antigen-binding portion comprises a different variable region thatrecognizes a different antigen, or a fragment or portion of the antibodythat binds to the two or more different antigens via its variableregions. As used herein, the term “bispecific antibody” refers to anantibody that comprises two different antigen-binding portions, in whicheach antigen-binding portion comprises a different variable region thatrecognizes a different antigen, or a fragment or portion of the antibodythat binds to the two different antigens via its variable regions.

A “monoclonal antibody” refers to antibodies produced by a single cloneof cells or a single cell line and consisting of or consistingessentially of antibody molecules that are identical in their primaryamino acid sequence.

A “polyclonal antibody” refers to an antibody obtained from aheterogeneous population of antibodies in which different antibodies inthe population bind to different epitopes of an antigen.

A “chimeric antibody” refers to an antibody molecule in which theconstant region, or a portion thereof, is altered, replaced or exchangedso that the antigen-binding site (i.e., variable region, CDR, or portionthereof) is linked to a constant region of a different or altered class,effector function and/or species, or in which the variable region, or aportion thereof, is altered, replaced or exchanged with a variableregion having a different or altered antigen specificity (e.g., CDR andframework regions from different species). In some embodiments, achimeric antibody is a monoclonal antibody comprising a variable regionfrom one source or species (e.g., mouse) and a constant region derivedfrom a second source or species (e.g., human). Methods for producingchimeric antibodies are described in the art.

A “humanized antibody” is a chimeric immunoglobulin derived from anon-human source (e.g., murine) that contains minimal sequences derivedfrom the non-human immunoglobulin outside the CDRs. In general, ahumanized antibody will comprise at least one (e.g., two)antigen-binding variable domain(s), in which the CDR regionssubstantially correspond to those of the non-human immunoglobulin andthe framework regions substantially correspond to those of a humanimmunoglobulin sequence. The humanized antibody can also comprise atleast a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin sequence. Methods of antibodyhumanization are known in the art.

A “human antibody” or a “fully human antibody” is an antibody havinghuman heavy chain and light chain sequences, typically derived fromhuman germline genes. In some embodiments, the antibody is produced by ahuman cell, by a non-human animal that utilizes human antibodyrepertoires (e.g., transgenic mice that are genetically engineered toexpress human antibody sequences), or by phage display platforms.

The term “specifically binds” refers to a molecule (e.g., an antibody oran antigen-binding portion thereof) that binds to an epitope or targetwith greater affinity, greater avidity, and/or greater duration to thatepitope or target in a sample than it binds to another epitope ornon-target compound (e.g., a structurally different antigen). In someembodiments, an antibody (or an antigen-binding portion thereof) thatspecifically binds to an epitope or target is an antibody (or anantigen-binding portion thereof) that binds to the epitope or targetwith at least 5-fold greater affinity than other epitopes or non-targetcompounds, e.g., at least 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 25-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or greateraffinity. The term “specific binding,” “specifically binds to,” or “isspecific for” a particular epitope or target, as used herein, can beexhibited, for example, by a molecule having an equilibrium dissociationconstant K_(D) for the epitope or target to which it binds of, e.g.,10⁻⁴ M or smaller, e.g., 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰M, 10⁻¹¹ M, or 10⁻¹² M. It will be recognized by one of skill that anantibody that specifically binds to a target (e.g., a TREM2 protein)from one species may also specifically bind to orthologs of that target(e.g., the TREM2 protein).

The term “binding affinity” is used herein to refer to the strength of anon-covalent interaction between two molecules, e.g., between anantibody (or an antigen-binding portion thereof) and an antigen. Thus,for example, the term may refer to 1:1 interactions between an antibody(or an antigen-binding portion thereof) and an antigen, unless otherwiseindicated or clear from context. Binding affinity may be quantified bymeasuring an equilibrium dissociation constant (K_(D)), which refers tothe dissociation rate constant (k_(d), time⁻¹) divided by theassociation rate constant (k_(a), time⁻¹ M⁻¹). K_(D) can be determinedby measurement of the kinetics of complex formation and dissociation,e.g., using Surface Plasmon Resonance (SPR) methods, e.g., a Biacore™system; kinetic exclusion assays such as KinExA®; and BioLayerinterferometry (e.g., using the ForteBio® Octet platform). As usedherein, “binding affinity” includes not only formal binding affinities,such as those reflecting 1:1 interactions between an antibody (or anantigen-binding portion thereof) and an antigen, but also apparentaffinities for which K_(D) values are calculated that may reflect avidbinding.

The term “cross-reacts,” as used herein, refers to the ability of anantibody to bind to an antigen other than the antigen against which theantibody was raised. In some embodiments, cross-reactivity refers to theability of an antibody to bind to an antigen from another species thanthe antigen against which the antibody was raised. As a non-limitingexample, an anti-TREM2 antibody as described herein that is raisedagainst a human TREM2 peptide can exhibit cross-reactivity with a TREM2peptide or protein from a different species (e.g., monkey or mouse).

The term “isolated,” as used with reference to a nucleic acid or protein(e.g., antibody), denotes that the nucleic acid or protein isessentially free of other cellular components with which it isassociated in the natural state. Purity and homogeneity are typicallydetermined using analytical chemistry techniques such as electrophoresis(e.g., polyacrylamide gel electrophoresis) or chromatography (e.g., highperformance liquid chromatography). In some embodiments, an isolatednucleic acid or protein (e.g., antibody) is at least 85% pure, at least90% pure, at least 95% pure, or at least 99% pure.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, y-carboxyglutamate, and O-phosphoserine. Naturallyoccurring a-amino acids include, without limitation, alanine (Ala),cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine(Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg),lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline(Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val),tryptophan (Trp), tyrosine (Tyr), and combinations thereof.Stereoisomers of a naturally occurring a-amino acids include, withoutlimitation, D-alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid(D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine(D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys),D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline(D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr),D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), andcombinations thereof “Amino acid analogs” refer to compounds that havethe same basic chemical structure as a naturally occurring amino acid,i.e., an a carbon that is bound to a hydrogen, a carboxyl group, anamino group, and an R group, e.g., homoserine, norleucine, methioninesulfoxide, methionine methyl sulfonium. Such analogs have modified Rgroups (e.g., norleucine) or modified peptide backbones, but retain thesame basic chemical structure as a naturally occurring amino acid.“Amino acid mimetics” refers to chemical compounds that have a structurethat is different from the general chemical structure of an amino acid,but that functions in a manner similar to a naturally occurring aminoacid. Amino acids may be referred to herein by either their commonlyknown three letter symbols or by the one-letter symbols recommended bythe IUPAC-IUB Biochemical Nomenclature Commission.

The terms “polypeptide” and “peptide” are used interchangeably herein torefer to a polymer of amino acid residues in a single chain. The termsapply to amino acid polymers in which one or more amino acid residue isan artificial chemical mimetic of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers andnon-naturally occurring amino acid polymers. Amino acid polymers maycomprise entirely L-amino acids, entirely D-amino acids, or a mixture ofL and D amino acids.

The term “protein” as used herein refers to either a polypeptide or adimer (i.e., two) or multimer (i.e., three or more) of single chainpolypeptides. The single chain polypeptides of a protein may be joinedby a covalent bond, e.g., a disulfide bond, or non-covalentinteractions.

The terms “polynucleotide” and “nucleic acid” interchangeably refer tochains of nucleotides of any length, and include DNA and RNA. Thenucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a chain by DNA or RNA polymerase. A polynucleotide maycomprise modified nucleotides, such as methylated nucleotides and theiranalogs. Examples of polynucleotides contemplated herein include single-and double-stranded DNA, single- and double-stranded RNA, and hybridmolecules having mixtures of single- and double-stranded DNA and RNA.

The terms “conservative substitution” and “conservative mutation” referto an alteration that results in the substitution of an amino acid withanother amino acid that can be categorized as having a similar feature.Examples of categories of conservative amino acid groups defined in thismanner can include: a “charged/polar group” including Glu (Glutamic acidor E), Asp (Aspartic acid or D), Asn (Asparagine or N), Gln (Glutamineor Q), Lys (Lysine or K), Arg (Arginine or R), and His (Histidine or H);an “aromatic group” including Phe (Phenylalanine or F), Tyr (Tyrosine orY), Trp (Tryptophan or W), and (Histidine or H); and an “aliphaticgroup” including Gly (Glycine or G), Ala (Alanine or A), Val (Valine orV), Leu (Leucine or L), Ile (Isoleucine or I), Met (Methionine or M),Ser (Serine or S), Thr (Threonine or T), and Cys (Cysteine or C). Withineach group, subgroups can also be identified. For example, the group ofcharged or polar amino acids can be sub-divided into sub-groupsincluding: a “positively-charged sub-group” comprising Lys, Arg and His;a “negatively-charged sub-group” comprising Glu and Asp; and a “polarsub-group” comprising Asn and Gln. In another example, the aromatic orcyclic group can be sub-divided into sub-groups including: a “nitrogenring sub-group” comprising Pro, His and Trp; and a “phenyl sub-group”comprising Phe and Tyr. In another further example, the aliphatic groupcan be sub-divided into sub-groups, e.g., an “aliphatic non-polarsub-group” comprising Val, Leu, Gly, and Ala; and an “aliphaticslightly-polar sub-group” comprising Met, Ser, Thr, and Cys. Examples ofcategories of conservative mutations include amino acid substitutions ofamino acids within the sub-groups above, such as, but not limited to:Lys for Arg or vice versa, such that a positive charge can bemaintained; Glu for Asp or vice versa, such that a negative charge canbe maintained; Ser for Thr or vice versa, such that a free —OH can bemaintained; and Gln for Asn or vice versa, such that a free —NH₂ can bemaintained. In some embodiments, hydrophobic amino acids are substitutedfor naturally occurring hydrophobic amino acid, e.g., in the activesite, to preserve hydrophobicity.

The terms “identical” or percent “identity,” in the context of two ormore polypeptide sequences, refer to two or more sequences orsubsequences that are the same or have a specified percentage of aminoacid residues, e.g., at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, or at least 95% orgreater, that are identical over a specified region when compared andaligned for maximum correspondence over a comparison window, ordesignated region as measured using a sequence comparison algorithm orby manual alignment and visual inspection.

For sequence comparison of polypeptides, typically one amino acidsequence acts as a reference sequence, to which a candidate sequence iscompared. Alignment can be performed using various methods available toone of skill in the art, e.g., visual alignment or using publiclyavailable software using known algorithms to achieve maximal alignment.Such programs include the BLAST programs, ALIGN, ALIGN-2 (Genentech,South San Francisco, Calif.) or Megalign (DNASTAR). The parametersemployed for an alignment to achieve maximal alignment can be determinedby one of skill in the art. For sequence comparison of polypeptidesequences for purposes of this application, the BLASTP algorithmstandard protein BLAST for aligning two proteins sequence with thedefault parameters is used.

The terms “subject,” “individual,” and “patient,” as usedinterchangeably herein, refer to a mammal, including but not limited tohumans, non-human primates, rodents (e.g., rats, mice, and guinea pigs),rabbits, cows, pigs, horses, and other mammalian species. In oneembodiment, the subject, individual, or patient is a human.

The terms “treating,” “treatment,” and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. “Treating” or “treatment” may refer to any indicia of success inthe treatment or amelioration of a neurodegenerative disease (e.g.,Alzheimer's disease or another neurodegenerative disease describedherein), including any objective or subjective parameter such asabatement, remission, improvement in patient survival, increase insurvival time or rate, diminishing of symptoms or making the diseasemore tolerable to the patient, slowing in the rate of degeneration ordecline, or improving a patient's physical or mental well-being. Thetreatment or amelioration of symptoms can be based on objective orsubjective parameters. The effect of treatment can be compared to anindividual or pool of individuals not receiving the treatment, or to thesame patient prior to treatment or at a different time during treatment.

The term “pharmaceutically acceptable excipient” refers to a non-activepharmaceutical ingredient that is biologically or pharmacologicallycompatible for use in humans or animals, such as, but not limited to abuffer, carrier, or preservative.

As used herein, a “therapeutic amount” or “therapeutically effectiveamount” of an agent (e.g., an antibody as described herein) is an amountof the agent that treats, alleviates, abates, or reduces the severity ofsymptoms of a disease in a subject. A “therapeutic amount” of an agent(e.g., an antibody as described herein) may improve patient survival,increase survival time or rate, diminish symptoms, make an injury,disease, or condition (e.g., a neurodegenerative disease) moretolerable, slow the rate of degeneration or decline, or improve apatient's physical or mental well-being.

The term “administer” refers to a method of delivering agents,compounds, or compositions to the desired site of biological action.These methods include, but are not limited to, topical delivery,parenteral delivery, intravenous delivery, intradermal delivery,intramuscular delivery, intrathecal delivery, colonic delivery, rectaldelivery, or intraperitoneal delivery. In one embodiment, an antibody asdescribed herein is administered intravenously.

The term “control” or “control value” refers to a reference value orbaseline value. Appropriate controls can be determined by one skilled inthe art. In some instances, control values can be determined relative toa baseline within the same subject or experiment, e.g., a measurement ofsTREM2 taken prior to treatment with an anti-TREM2 antibody can be acontrol value for a post-treatment measurement of sTREM2 levels in thesame subject. In other instances, the control value can be determinedrelative to a control subject (e.g., a healthy control or a diseasecontrol) or an average value in a population of control subjects (e.g.,healthy controls or disease controls, e.g., a population of 10, 20, 50,100, 200, 500, 1000 control subjects or more), e.g, a measurement of asubject's level of sTREM2 either at baseline or after treatment can becompared to a healthy control value.

III. Anti-Trem2 Antibodies

In one aspect, antibodies and antigen-binding fragments thereof thatspecifically bind to a TREM2 protein are provided. In some embodiments,the antibody specifically binds to a human TREM2 protein. In someembodiments, an anti-TREM2 antibody is selective for TREM2 over otherTREM-like receptors (e.g., TREM1).

In some embodiments, an anti-TREM2 antibody is an antibody thatcomprises one or more complementarity determining region (CDR), heavychain variable region, and/or light chain variable region sequences asdisclosed herein. In some embodiments, an anti-TREM2 antibody comprisesone or more CDR, heavy chain variable region, and/or light chainvariable region sequences as disclosed herein and further comprises oneor more functional characteristics as disclosed herein, e.g., anantibody that enhances TREM2 activity (e.g., enhances phagocytosis, orenhances the migration, differentiation, function, or survival of a cellsuch as a myeloid cell, microglia, or macrophage) or an antibody thatdecreases levels of sTREM2.

Anti-TREM2 Antibody Sequences

In some embodiments, an anti-TREM2 or antigen-binding fragment thereofcomprises a heavy chain sequence, or a portion thereof, and/or a lightchain sequence, or a portion thereof, derived from any of the followinganti-TREM2 antibodies described herein: Clone CL0020306, CloneCL0020188, Clone CL0020307, and Clone CL0020123. The CDR, heavy chainvariable region, and light chain variable region amino acid sequences ofthese clones is set forth in the Informal Sequence Listing. In someembodiments, the anti-TREM2 antibody is a chimeric antibody. In someembodiments, the anti-TREM2 antibody is a humanized and/or affinitymatured antibody.

In some embodiments, an anti-TREM2 antibody comprises one or more CDRsselected from the group consisting of:

-   -   (a) a heavy chain CDR1 (CDR-H1) sequence having at least 90%        sequence identity to the amino acid sequence of any one of SEQ        ID NOS:4, 12, and 29 or having up to two amino acid        substitutions relative to the amino acid sequence of any one of        SEQ ID NOS:4, 12, and 29;    -   (b) a heavy chain CDR2 (CDR-H2) sequence having at least 90%        sequence identity to the amino acid sequence of any one of SEQ        ID NOS:5, 13, 25, 30, 39, 41, and 43 or having up to two amino        acid substitutions relative to the amino acid sequence of any        one of SEQ ID NOS:5, 13, 25, 30, 39, 41, and 43;    -   (c) a heavy chain CDR3 (CDR-H3) sequence having at least 90%        sequence identity to the amino acid sequence of any one of SEQ        ID NOS:6, 14, 17, and 31 or having up to two amino acid        substitutions relative to the amino acid sequence of any one of        SEQ ID NOS:6, 14, 17, and 31;    -   (d) a light chain CDR1 (CDR-L1) sequence having at least 90%        sequence identity to the amino acid sequence of any one of SEQ        ID NOS:7, 23, and 32 or having up to two amino acid        substitutions relative to the amino acid sequence of any one of        SEQ ID NOS:7, 23, and 32;    -   (e) a light chain CDR2 (CDR-L2) sequence having at least 90%        sequence identity to the amino acid sequence of any one of SEQ        ID NOS:8 and 33 or having up to two amino acid substitutions        relative to the amino acid sequence of any one of SEQ ID NOS:8        and 33; and    -   (f) a light chain CDR3 (CDR-L3) sequence having at least 90%        sequence identity to the amino acid sequence of any one of SEQ        ID NOS:9, 18, and 34 or having up to two amino acid        substitutions relative to the amino acid sequence of any one of        SEQ ID NOS:9, 18, and 34.

In some embodiments, an anti-TREM2 antibody comprises two, three, four,five, or all six of (a)-(f). In some embodiments, an anti-TREM2 antibodycomprises the CDR-H1 of (a), the CDR-H2 of (b), and the CDR-H3 of (c).In some embodiments, an anti-TREM2 antibody comprises the CDR-L1 of (d),the CDR-L2 of (e), and the CDR-L3 of (f). In some embodiments, a CDRhaving up to two amino acid substitutions has one amino acidsubstitution relative to the reference sequence. In some embodiments, aCDR having up to two amino acid substitutions has two amino acidsubstitutions relative to the reference sequence. In some embodiments,the up to two amino acid substitutions are conservative substitutions.

In some embodiments, an anti-TREM2 antibody comprises one or more CDRsselected from the group consisting of:

-   -   (a) a CDR-H1 sequence comprising the amino acid sequence of any        one of SEQ ID NOS:4, 12, and 29;    -   (b) a CDR-H2 sequence comprising the amino acid sequence of any        one of SEQ ID NOS:5, 13, 25, 30, 39, 41, and 43;    -   (c) a CDR-H3 sequence comprising the amino acid sequence of any        one of SEQ ID NOS:6, 14, 17, and 31;    -   (d) a CDR-L1 sequence comprising the amino acid sequence of any        one of SEQ ID NOS:7, 23, and 32;    -   (e) a CDR-L2 sequence comprising the amino acid sequence of any        one of SEQ ID NOS:8 and 33; and    -   (f) a CDR-L3 sequence comprising the amino acid sequence of any        one of SEQ ID NOS:9, 18, and 34.

In some embodiments, an anti-TREM2 antibody comprises two, three, four,five, or all six of (a)-(f). In some embodiments, an anti-TREM2 antibodycomprises the CDR-H1 of

-   -   (a), the CDR-H2 of (b), and the CDR-H3 of (c). In some        embodiments, an anti-TREM2 antibody comprises the CDR-L1 of (d),        the CDR-L2 of (e), and the CDR-L3 of (f).

In some embodiments, an anti-TREM2 antibody comprises:

-   -   (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:5, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:6, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:9; or    -   (b) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:5, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:17, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:18; or    -   (c) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:5, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:17, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:23, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:18; or    -   (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:25, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:17, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:18; or    -   (e) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:25, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:17, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:23, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:18; or    -   (f) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:12,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:13, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:14, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:9; or    -   (g) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:29,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:30, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:31, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:32, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:33, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:34; or    -   (h) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:29,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:39, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:31, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:32, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:33, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:34; or    -   (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:29,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:41, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:31, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:32, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:33, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:34; or    -   (j) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:29,        a CDR-H2 comprising the amino acid sequence of SEQ ID NO:43, a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:31, a        CDR-L1 comprising the amino acid sequence of SEQ ID NO:32, a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:33, and a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:34.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region comprising an amino acid sequence that has at least 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity toany one of SEQ ID NOS:2, 10, 15, 19, 21, 24, 26, 27, 35, 37, 38, 40, 42,44, 45, 46, and 79. In some embodiments, an anti-TREM2 comprises a heavychain variable region comprising the amino acid sequence of any one ofSEQ ID NOS:2, 10, 15, 19, 21, 24, 26, 27, 35, 37, 38, 40, 42, 44, 45,46, and 79.

In some embodiments, an anti-TREM2 antibody comprises a light chainvariable region comprising an amino acid sequence that has at least 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity toany one of SEQ ID NOS:3, 11, 16, 20, 22, 28, 36, and 68. In someembodiments, an anti-TREM2 antibody comprises a light chain variableregion comprising the amino acid sequence of any one of SEQ ID NOS:3,11, 16, 20, 22, 28, 36, and 68.

In some embodiments, an anti-TREM2 antibody comprises: a heavy chainvariable region comprising an amino acid sequence that has at least 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity toany one of SEQ ID NOS:2, 10, 15, 19, 21, 24, 26, 27, 35, 37, 38, 40, 42,44, 45, 46, and 79, and a light chain variable region comprising anamino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOS:3, 11,16, 20, 22, 28, 36, and 68. In some embodiments, an anti-TREM2comprises: a heavy chain variable region comprising the amino acidsequence of any one of SEQ ID NOS:2, 10, 15, 19, 21, 24, 26, 27, 35, 37,38, 40, 42, 44, 45, 46, and 79, and a light chain variable regioncomprising the amino acid sequence of any one of SEQ ID NOS:3, 11, 16,20, 22, 28, 36, and 68.

In some embodiments, an anti-TREM2 antibody comprises:

-   -   (a) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:2 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:3; or    -   (b) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:10 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:11; or    -   (c) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:15 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:16; or    -   (d) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:19 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:20; or    -   (e) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:21 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:20; or    -   (f) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:19 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:22; or    -   (g) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:79 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:22; or    -   (h) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:24 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:20; or    -   (i) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:26 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:20; or    -   (j) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:24 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:22; or    -   (k) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:26 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:22; or    -   (l) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:27 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:28; or    -   (m) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:35 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:36; or    -   (n) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:37 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:36; or    -   (o) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:38 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:36; or    -   (p) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:40 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:36; or    -   (q) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:42 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:36; or    -   (r) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:44 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:36; or    -   (s) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:45 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:36; or    -   (t) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:46 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:36; or    -   (u) a V_(H) sequence that has at least 85% sequence identity to        SEQ ID NO:24 and a V_(L) sequence that has at least 85% sequence        identity to SEQ ID NO:68.

In some embodiments, an anti-TREM2 antibody comprises one or moresequences that are encompassed by a consensus sequence disclosed herein.As a non-limiting example, consensus sequences can be identified byaligning heavy chain or light chain sequences (e.g., CDRs) forantibodies that are from the same (or similar) germlines. In someembodiments, consensus sequences may be generated from antibodies thatcontain sequences that are of the same (or similar) length and/or haveat least one highly similar CDR (e.g., a highly similar CDR3). In someembodiments, such sequences in these antibodies may be aligned andcompared to identify conserved amino acids or motifs (i.e., wherealteration in sequences may alter protein function) and/or regions wherevariation occurs the sequences (i.e., where variation of sequence is notlikely to significantly affect protein function). Alternatively,consensus sequences can be identified by aligning heavy chain or lightchain sequences (e.g., CDRs) for antibodies that bind to the same orsimilar (e.g., overlapping) epitopes to determine conserved amino acidsor motifs (i.e., where alteration in sequences may alter proteinfunction) and regions where variation occurs in alignment of sequences(i.e., where variation of sequence is not likely to significantly affectprotein function). In some embodiments, one or more consensus sequencescan be identified for antibodies that recognize the same or similarepitope as an anti-TREM2 antibody as disclosed herein. Exemplaryconsensus sequences include SEQ ID NOS:47-52. In the consensus sequencesof SEQ ID NOS:47-52, the capitalized letter represents an amino acidresidue that is absolutely conserved among the aligned sequences (e.g.,aligned CDR sequences), while an “X” or a Greek letter (e.g., “α,” “β,”“γ,” “δ,” “ε,” or “φ”) represents an amino acid residue that is notabsolutely conserved among the aligned sequences. It will be appreciatedthat, when selecting an amino acid to insert at a position marked by an“X” or by a Greek letter, in some embodiments the amino acid is selectedfrom those amino acids found at the corresponding position in thealigned sequences.

Clone CL0020123 and Variants of CL0020123

In some embodiments, an anti-TREM2 antibody or antigen-binding fragmentthereof comprises:

-   -   (a) a CDR-H1 sequence comprising the sequence of GFSIEDFYIH (SEQ        ID NO:29);    -   (b) a CDR-H2 sequence comprising the sequence of        W-I-D-P-E-β₆-G-β₈-S—K-Y-A-P—K-F-Q-G (SEQ ID NO:47), wherein β₆        is N or Q and β₈ is D or E;    -   (c) a CDR-H3 sequence comprising the sequence of HADHGNYGSTMDY        (SEQ ID NO:31);    -   (d) a CDR-L1 sequence comprising the sequence of HASQHINVWLS        (SEQ ID NO:32);    -   (e) a CDR-L2 sequence comprising the sequence of KASNLHT (SEQ ID        NO:33); and    -   (f) a CDR-L3 sequence comprising the sequence of QQGQTYPRT (SEQ        ID NO:34).

In some embodiments, an anti-TREM2 antibody comprises a CDR-H2 sequencethat is selected from SEQ ID NOS:30, 39, 41, and 43.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to any one of SEQ ID NOS:27, 35, 37,38, 40, 42, 44, 45, and 46. In some embodiments, an anti-TREM2 antibodycomprises a heavy chain variable region comprising the amino acidsequence of any one of SEQ ID NOS:27, 35, 37, 38, 40, 42, 44, 45, and46.

In some embodiments, an anti-TREM2 antibody comprises a light chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to any one of SEQ ID NOS:28 and 36.In some embodiments, an anti-TREM2 antibody comprises a light chainvariable region comprising the amino acid sequence of any one of SEQ IDNOS:28 and 36.

In some embodiments, an anti-TREM2 antibody comprises a CDR-H1 sequencecomprising the amino acid sequence of SEQ ID NO:29, a CDR-H2 sequencecomprising the amino acid sequence of SEQ ID NO:30, a CDR-H3 sequencecomprising the amino acid sequence of SEQ ID NO:31, a CDR-L1 sequencecomprising the amino acid sequence of SEQ ID NO:32, a CDR-L2 sequencecomprising the amino acid sequence of SEQ ID NO:33, and a CDR-L3sequence comprising the amino acid sequence of SEQ ID NO:34.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to SEQ ID NO:27. In someembodiments, an anti-TREM2 antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:27.

In some embodiments, an anti-TREM2 antibody comprises a light chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to SEQ ID NO:28. In someembodiments, an anti-TREM2 antibody comprises a light chain variableregion comprising the amino acid sequence of SEQ ID NO:28.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to SEQ ID NO:27 and a light chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to SEQ ID NO:28. In someembodiments, an anti-TREM2 antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:27 and a lightchain variable region comprising the amino acid sequence of SEQ IDNO:28.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region that comprises a heavy chain CDR1-3 comprising the aminoacid sequences of SEQ ID NOS:29, 30, and 31, respectively, and that hasat least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:27. In someembodiments, an anti-TREM2 antibody comprises a light chain variableregion that comprises a light chain CDR1-3 comprising the amino acidsequences of SEQ ID NOS:32, 33, and 34, respectively, and that has atleast 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:28.

In some embodiments, an anti-TREM2 antibody is an antibody that competesfor binding with an antibody as described herein (e.g., an antibodycomprising a heavy chain CDR1-3 and a light chain CDR1-3 comprising theamino acid sequences of SEQ ID NOS:29, 30, 31, 32, 33, and 34,respectively, or an antibody comprising a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:27 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO:28).

In some embodiments, an anti-TREM2 antibody comprises a CDR-H1 sequencecomprising the amino acid sequence of SEQ ID NO:29, a CDR-H2 sequencecomprising the amino acid sequence of SEQ ID NO:43, a CDR-H3 sequencecomprising the amino acid sequence of SEQ ID NO:31, a CDR-L1 sequencecomprising the amino acid sequence of SEQ ID NO:32, a CDR-L2 sequencecomprising the amino acid sequence of SEQ ID NO:33, and a CDR-L3sequence comprising the amino acid sequence of SEQ ID NO:34.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) toSEQ ID NO:42. In some embodiments, an anti-TREM2 antibody comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO:42.

In some embodiments, an anti-TREM2 antibody comprises a light chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) toSEQ ID NO:36. In some embodiments, an anti-TREM2 antibody comprises alight chain variable region comprising the amino acid sequence of SEQ IDNO:36.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) toSEQ ID NO:42 and a light chain variable region comprising an amino acidsequence that has at least 85% sequence identity (e.g., at least 90%,95%, or 97% sequence identity) to SEQ ID NO:36. In some embodiments, ananti-TREM2 antibody comprises a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO:42 and a light chain variableregion comprising the amino acid sequence of SEQ ID NO:36.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region that comprises a heavy chain CDR1-3 comprising the aminoacid sequences of SEQ ID NOs:29, 43, and 31, respectively, and that hasat least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequenceidentity) to SEQ ID NO:42. In some embodiments, an anti-TREM2 antibodycomprises a light chain variable region that comprises a light chainCDR1-3 comprising the amino acid sequences of SEQ ID NOs:32, 33, and 34,respectively, and that has at least 85% sequence identity (e.g., atleast 90%, 95%, or 97% sequence identity) to SEQ ID NO:36.

In some embodiments, an anti-TREM2 antibody is an antibody that competesfor binding with an antibody as described herein (e.g., an antibodycomprising a heavy chain CDR1-3 and a light chain CDR1-3 comprising theamino acid sequences of SEQ ID NOS:29, 43, 31, 32, 33, and 34,respectively, or an antibody comprising a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:42 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO:36).

In some embodiments, an anti-TREM2 antibody comprises a CDR-H1 sequencecomprising the amino acid sequence of SEQ ID NO:29, a CDR-H2 sequencecomprising the amino acid sequence of SEQ ID NO:41, a CDR-H3 sequencecomprising the amino acid sequence of SEQ ID NO:31, a CDR-L1 sequencecomprising the amino acid sequence of SEQ ID NO:32, a CDR-L2 sequencecomprising the amino acid sequence of SEQ ID NO:33, and a CDR-L3sequence comprising the amino acid sequence of SEQ ID NO:34.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) toSEQ ID NO:45. In some embodiments, an anti-TREM2 antibody comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO:45.

In some embodiments, an anti-TREM2 antibody comprises a light chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) toSEQ ID NO:36. In some embodiments, an anti-TREM2 antibody comprises alight chain variable region comprising the amino acid sequence of SEQ IDNO:36.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) toSEQ ID NO:45 and a light chain variable region comprising an amino acidsequence that has at least 85% sequence identity (e.g., at least 90%,95%, or 97% sequence identity) to SEQ ID NO:36. In some embodiments, ananti-TREM2 antibody comprises a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO:45 and a light chain variableregion comprising the amino acid sequence of SEQ ID NO:36.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region that comprises a heavy chain CDR1-3 comprising the aminoacid sequences of SEQ ID NOs:29, 41, and 31, respectively, and that hasat least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequenceidentity) to SEQ ID NO:45. In some embodiments, an anti-TREM2 antibodycomprises a light chain variable region that comprises a light chainCDR1-3 comprising the amino acid sequences of SEQ ID NOs:32, 33, and 34,respectively, and that has at least 85% sequence identity (e.g., atleast 90%, 95%, or 97% sequence identity) to SEQ ID NO:36.

In some embodiments, an anti-TREM2 antibody is an antibody that competesfor binding with an antibody as described herein (e.g., an antibodycomprising a heavy chain CDR1-3 and a light chain CDR1-3 comprising theamino acid sequences of SEQ ID NOS:29, 41, 31, 32, 33, and 34,respectively, or an antibody comprising a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:45 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO:36).

Clones CL0020188, CL0020306, CL0020307, and Variants of CL0020188

In some embodiments, an anti-TREM2 antibody or antigen-binding fragmentthereof comprises:

-   -   (a) a CDR-H1 sequence comprising the sequence of        G-F-T-F-T-α₆-F-Y-M-S(SEQ ID NO:48), wherein α₆ is D or N;    -   (b) a CDR-H2 sequence comprising the sequence of        V—I-R-N-β₅-β₆-N-β₈-Y-T-β₁₁-β₁₂-Y—N-P-S—V-K-G (SEQ ID NO:49),        wherein 3₅ is K or R; β₆ is A or P; β₈ is G or A; β₁₁ is A or T;        and β₁₂ is G or D;    -   (c) a CDR-H3 sequence comprising the sequence of        γ₁-R-L-γ₄-Y-G-F-D-Y (SEQ ID NO:50), wherein γ₁ is A or T; and γ₄        is T or S;    -   (d) a CDR-L1 sequence comprising the sequence of        Q-S—S-K-S-L-L-H-S-(SEQ ID NO:51), wherein δ₁₀ is N or T;    -   (e) a CDR-L2 sequence comprising the sequence of WMSTRAS (SEQ ID        NO:8); and    -   (f) a CDR-L3 sequence comprising the sequence of        Q-Q-F-L-E-ϕ₆-P-F-T (SEQ ID NO:52), wherein ϕ₆ is Y or F.

In some embodiments, an anti-TREM2 antibody comprises a CDR-H1 sequencethat is selected from SEQ ID NOS:4 and 12. In some embodiments, ananti-TREM2 antibody comprises a CDR-H2 sequence that is selected fromSEQ ID NOS:5, 13, and 25. In some embodiments, an anti-TREM2 antibodycomprises a CDR-H3 sequence that is selected from SEQ ID NOS:6, 14, and17. In some embodiments, an anti-TREM2 antibody comprises a CDR-L1sequence that is selected from SEQ ID NOS:7 and 23. In some embodiments,an anti-TREM2 antibody comprises a CDR-L3 sequence is selected from SEQID NOS:9 and 18.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to any one of SEQ ID NOS:2, 10, 15,19, 21, 24, 26, and 79. In some embodiments, an anti-TREM2 antibodycomprises a heavy chain variable region comprising the amino acidsequence of any one of SEQ ID NOS:2, 10, 15, 19, 21, 24, 26, and 79.

In some embodiments, an anti-TREM2 antibody comprises a light chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to any one of SEQ ID NOS:3, 11, 16,20, 22, and 68. In some embodiments, an anti-TREM2 antibody comprises alight chain variable region comprising the amino acid sequence of anyone of SEQ ID NOS:3, 11, 16, 20, 22, and 68.

Clone CL0020188 and Variants of CL0020188

In some embodiments, an anti-TREM2 antibody comprises a CDR-H1 sequencecomprising the amino acid sequence of SEQ ID NO:4, a CDR-H2 sequencecomprising the amino acid sequence of SEQ ID NO:5, a CDR-H3 sequencecomprising the amino acid sequence of SEQ ID NO:17, a CDR-L1 sequencecomprising the amino acid sequence of SEQ ID NO:7, a CDR-L2 sequencecomprising the amino acid sequence of SEQ ID NO:8, and a CDR-L3 sequencecomprising the amino acid sequence of SEQ ID NO:18.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to SEQ ID NO:15. In someembodiments, an anti-TREM2 antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:15.

In some embodiments, an anti-TREM2 antibody comprises a light chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to SEQ ID NO:16. In someembodiments, an anti-TREM2 antibody comprises a light chain variableregion comprising the amino acid sequence of SEQ ID NO:16.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to SEQ ID NO:15 and a light chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to SEQ ID NO:16. In someembodiments, an anti-TREM2 antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:15 and a lightchain variable region comprising the amino acid sequence of SEQ IDNO:16.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region that comprises a heavy chain CDR1-3 comprising the aminoacid sequences of SEQ ID NOS:4, 5, and 17, respectively, and that has atleast 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:15. In someembodiments, an anti-TREM2 antibody comprises a light chain variableregion that comprises a light chain CDR1-3 comprising the amino acidsequences of SEQ ID NOS:7, 8, and 18, respectively, and that has atleast 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:16.

In some embodiments, an anti-TREM2 antibody is an antibody that competesfor binding with an antibody as described herein (e.g., an antibodycomprising a heavy chain CDR1-3 and a light chain CDR1-3 comprising theamino acid sequences of SEQ ID NOS:5, 17, 7, 8, and 18, respectively, oran antibody comprising a heavy chain variable region comprising theamino acid sequence of SEQ ID NO:15 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:16).

In some embodiments, an anti-TREM2 antibody comprises a CDR-H1 sequencecomprising the amino acid sequence of SEQ ID NO:4, a CDR-H2 sequencecomprising the amino acid sequence of SEQ ID NO:5, a CDR-H3 sequencecomprising the amino acid sequence of SEQ ID NO:17, a CDR-L1 sequencecomprising the amino acid sequence of SEQ ID NO:23, a CDR-L2 sequencecomprising the amino acid sequence of SEQ ID NO:8, and a CDR-L3 sequencecomprising the amino acid sequence of SEQ ID NO:18.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) toSEQ ID NO:79. In some embodiments, an anti-TREM2 antibody comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO:79.

In some embodiments, an anti-TREM2 antibody comprises a light chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) toSEQ ID NO:22. In some embodiments, an anti-TREM2 antibody comprises alight chain variable region comprising the amino acid sequence of SEQ IDNO:22.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) toSEQ ID NO:79 and a light chain variable region comprising an amino acidsequence that has at least 85% sequence identity (e.g., at least 90%,95%, or 97% sequence identity) to SEQ ID NO:22. In some embodiments, ananti-TREM2 antibody comprises a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO:79 and a light chain variableregion comprising the amino acid sequence of SEQ ID NO:22.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region that comprises a heavy chain CDR1-3 comprising the aminoacid sequences of SEQ ID NOs:4, 5, and 17, respectively, and that has atleast 85% sequence identity (e.g., at least 90%, 95%, or 97% sequenceidentity) to SEQ ID NO:79. In some embodiments, an anti-TREM2 antibodycomprises a light chain variable region that comprises a light chainCDR1-3 comprising the amino acid sequences of SEQ ID NOs:23, 8, and 18,respectively, and that has at least 85% sequence identity (e.g., atleast 90%, 95%, or 97% sequence identity) to SEQ ID NO:22.

In some embodiments, an anti-TREM2 antibody is an antibody that competesfor binding with an antibody as described herein (e.g., an antibodycomprising a heavy chain CDR1-3 and a light chain CDR1-3 comprising theamino acid sequences of SEQ ID NOS:4, 5, 17, 23, 8, and 18,respectively, or an antibody comprising a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:79 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO:22).

In some embodiments, an anti-TREM2 antibody comprises a CDR-H1 sequencecomprising the amino acid sequence of SEQ ID NO:4, a CDR-H2 sequencecomprising the amino acid sequence of SEQ ID NO:25, a CDR-H3 sequencecomprising the amino acid sequence of SEQ ID NO:17, a CDR-L1 sequencecomprising the amino acid sequence of SEQ ID NO:23, a CDR-L2 sequencecomprising the amino acid sequence of SEQ ID NO:8, and a CDR-L3 sequencecomprising the amino acid sequence of SEQ ID NO:18.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) toSEQ ID NO:24. In some embodiments, an anti-TREM2 antibody comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO:24.

In some embodiments, an anti-TREM2 antibody comprises a light chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) toSEQ ID NO:22. In some embodiments, an anti-TREM2 antibody comprises alight chain variable region comprising the amino acid sequence of SEQ IDNO:22.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) toSEQ ID NO:24 and a light chain variable region comprising an amino acidsequence that has at least 85% sequence identity (e.g., at least 90%,95%, or 97% sequence identity) to SEQ ID NO:22. In some embodiments, ananti-TREM2 antibody comprises a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO:24 and a light chain variableregion comprising the amino acid sequence of SEQ ID NO:22.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region that comprises a heavy chain CDR1-3 comprising the aminoacid sequences of SEQ ID NOs:4, 25, and 17, respectively, and that hasat least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequenceidentity) to SEQ ID NO:24. In some embodiments, an anti-TREM2 antibodycomprises a light chain variable region that comprises a light chainCDR1-3 comprising the amino acid sequences of SEQ ID NOs:23, 8, and 18,respectively, and that has at least 85% sequence identity (e.g., atleast 90%, 95%, or 97% sequence identity) to SEQ ID NO:22.

In some embodiments, an anti-TREM2 antibody is an antibody that competesfor binding with an antibody as described herein (e.g., an antibodycomprising a heavy chain CDR1-3 and a light chain CDR1-3 comprising theamino acid sequences of SEQ ID NOS:4, 25, 17, 23, 8, and 18,respectively, or an antibody comprising a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:24 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO:22).

In some embodiments, an anti-TREM2 antibody comprises a CDR-H1 sequencecomprising the amino acid sequence of SEQ ID NO:4, a CDR-H2 sequencecomprising the amino acid sequence of SEQ ID NO:25, a CDR-H3 sequencecomprising the amino acid sequence of SEQ ID NO:17, a CDR-L1 sequencecomprising the amino acid sequence of SEQ ID NO:7, a CDR-L2 sequencecomprising the amino acid sequence of SEQ ID NO:8, and a CDR-L3 sequencecomprising the amino acid sequence of SEQ ID NO:9.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) toSEQ ID NO:24. In some embodiments, an anti-TREM2 antibody comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO:24.

In some embodiments, an anti-TREM2 antibody comprises a light chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) toSEQ ID NO:68. In some embodiments, an anti-TREM2 antibody comprises alight chain variable region comprising the amino acid sequence of SEQ IDNO:68.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 95%, or 97% sequence identity) toSEQ ID NO:24 and a light chain variable region comprising an amino acidsequence that has at least 85% sequence identity (e.g., at least 90%,95%, or 97% sequence identity) to SEQ ID NO:68. In some embodiments, ananti-TREM2 antibody comprises a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO:24 and a light chain variableregion comprising the amino acid sequence of SEQ ID NO:68.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region that comprises a heavy chain CDR1-3 comprising the aminoacid sequences of SEQ ID NOs:4, 25, and 17, respectively, and that hasat least 85% sequence identity (e.g., at least 90%, 95%, or 97% sequenceidentity) to SEQ ID NO:24. In some embodiments, an anti-TREM2 antibodycomprises a light chain variable region that comprises a light chainCDR1-3 comprising the amino acid sequences of SEQ ID NOs:7, 8, and 9,respectively, and that has at least 85% sequence identity (e.g., atleast 90%, 95%, or 97% sequence identity) to SEQ ID NO:68.

In some embodiments, an anti-TREM2 antibody is an antibody that competesfor binding with an antibody as described herein (e.g., an antibodycomprising a heavy chain CDR1-3 and a light chain CDR1-3 comprising theamino acid sequences of SEQ ID NOS:4, 25, 17, 7, 8, and 9, respectively,or an antibody comprising a heavy chain variable region comprising theamino acid sequence of SEQ ID NO:24 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:68).

Clone CL0020306

In some embodiments, an anti-TREM2 antibody comprises a CDR-H1 sequencecomprising the amino acid sequence of SEQ ID NO:4, a CDR-H2 sequencecomprising the amino acid sequence of SEQ ID NO:5, a CDR-H3 sequencecomprising the amino acid sequence of SEQ ID NO:6, a CDR-L1 sequencecomprising the amino acid sequence of SEQ ID NO:7, a CDR-L2 sequencecomprising the amino acid sequence of SEQ ID NO:8, and a CDR-L3 sequencecomprising the amino acid sequence of SEQ ID NO:9.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to SEQ ID NO:2. In some embodiments,an anti-TREM2 antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:2.

In some embodiments, an anti-TREM2 antibody comprises a light chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to SEQ ID NO:3. In some embodiments,an anti-TREM2 antibody comprises a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:3.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to SEQ ID NO:2 and a light chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to SEQ ID NO:3. In some embodiments,an anti-TREM2 antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:2 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO:3.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region that comprises a heavy chain CDR1-3 comprising the aminoacid sequences of SEQ ID NOS:4, 5, and 6, respectively, and that has atleast 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:2. In someembodiments, an anti-TREM2 antibody comprises a light chain variableregion that comprises a light chain CDR1-3 comprising the amino acidsequences of SEQ ID NOS:7, 8, and 9, respectively, and that has at least85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to SEQ ID NO:3.

In some embodiments, an anti-TREM2 antibody is an antibody that competesfor binding with an antibody as described herein (e.g., an antibodycomprising a heavy chain CDR1-3 and a light chain CDR1-3 comprising theamino acid sequences of SEQ ID NOS:4, 5, 6, 7, 8, and 9, respectively,or an antibody comprising a heavy chain variable region comprising theamino acid sequence of SEQ ID NO:2 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:3).

Clone CL0020307

In some embodiments, an anti-TREM2 antibody comprises a CDR-H1 sequencecomprising the amino acid sequence of SEQ ID NO:12, a CDR-H2 sequencecomprising the amino acid sequence of SEQ ID NO:13, a CDR-H3 sequencecomprising the amino acid sequence of SEQ ID NO:14, a CDR-L1 sequencecomprising the amino acid sequence of SEQ ID NO:7, a CDR-L2 sequencecomprising the amino acid sequence of SEQ ID NO:8, and a CDR-L3 sequencecomprising the amino acid sequence of SEQ ID NO:9.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to SEQ ID NO:10. In someembodiments, an anti-TREM2 antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:10.

In some embodiments, an anti-TREM2 antibody comprises a light chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to SEQ ID NO:11. In someembodiments, an anti-TREM2 antibody comprises a light chain variableregion comprising the amino acid sequence of SEQ ID NO:11.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to SEQ ID NO:10 and a light chainvariable region comprising an amino acid sequence that has at least 85%sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to SEQ ID NO:11. In someembodiments, an anti-TREM2 antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:10 and a lightchain variable region comprising the amino acid sequence of SEQ IDNO:11.

In some embodiments, an anti-TREM2 antibody comprises a heavy chainvariable region that comprises a heavy chain CDR1-3 comprising the aminoacid sequences of SEQ ID NOS:12, 13, and 14, respectively, and that hasat least 85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO:10. In someembodiments, an anti-TREM2 antibody comprises a light chain variableregion that comprises a light chain CDR1-3 comprising the amino acidsequences of SEQ ID NOS:7, 8, and 9, respectively, and that has at least85% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to SEQ ID NO:11.

In some embodiments, an anti-TREM2 antibody is an antibody that competesfor binding with an antibody as described herein (e.g., an antibodycomprising a heavy chain CDR1-3 and a light chain CDR1-3 comprising theamino acid sequences of SEQ ID NOS:12, 13, 14, 7, 8, and 9,respectively, or an antibody comprising a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:10 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO:11).

Binding Characteristics of Anti-TREM2 Antibodies

In some embodiments, an antibody as described herein that specificallybinds to a TREM2 protein binds to TREM2 that is expressed on a cell(e.g., a primary cell or cell line that endogenously expresses TREM2,such as human macrophages, or a primary cell or cell line that has beenengineered to express TREM2, e.g., as described in the Examples sectionbelow). In some embodiments, an antibody that specifically binds to aTREM2 protein as described herein binds to purified or recombinant TREM2protein of a portion thereof, or to a chimeric protein comprising TREM2or a portion thereof (e.g., an Fc-fusion protein comprising TREM2 or anFc-fusion protein comprising the ecto-domain of TREM2).

In some embodiments, an antibody that specifically binds to human TREM2protein exhibits cross-reactivity with one or more other TREM2 proteinsof another species. In some embodiments, an antibody that specificallybinds to human TREM2 protein exhibits cross-reactivity with a cynomolgusmonkey (“cyno”) TREM2 protein. In some embodiments, an antibody thatspecifically binds to human TREM2 protein exhibits cross-reactivity witha mouse TREM2 protein. In some embodiments, an anti-TREM2 antibodyexhibits cross-reactivity with human TREM2, cyno TREM2, and mouse TREM2.

Methods for analyzing binding affinity, binding kinetics, andcross-reactivity are known in the art. These methods include, but arenot limited to, solid-phase binding assays (e.g., ELISA assay),immunoprecipitation, surface plasmon resonance (e.g., Biacore™ (GEHealthcare, Piscataway, N.J.)), kinetic exclusion assays (e.g.,KinExA®), flow cytometry, fluorescence-activated cell sorting (FACS),BioLayer interferometry (e.g., Octet™ (FortéBio, Inc., Menlo Park,Calif.)), and western blot analysis. In some embodiments, ELISA is usedto determine binding affinity and/or cross-reactivity. Methods forperforming ELISA assays are known in the art, and are also described inthe Examples section below. In some embodiments, surface plasmonresonance (SPR) is used to determine binding affinity, binding kinetics,and/or cross-reactivity. In some embodiments, kinetic exclusion assaysare used to determine binding affinity, binding kinetics, and/orcross-reactivity. In some embodiments, BioLayer interferometry assaysare used to determine binding affinity, binding kinetics, and/orcross-reactivity.

Epitopes Recognized by Anti-TREM2 Antibodies

In some embodiments, an anti-TREM2 antibody recognizes an epitope ofhuman TREM2 that is the same or substantially the same as the epitoperecognized by an antibody clone as described herein. As used herein, theterm “substantially the same,” as used with reference to an epitoperecognized by an antibody clone as described herein, means that theanti-TREM2 antibody recognizes an epitope that is identical, within, ornearly identical to (e.g., has at least 90% sequence identity to, or hasone, two, or three amino acid substitutions, e.g., conservativesubstitutions, relative to), or has substantial overlap with (e.g., atleast 50%, 60%, 70%, 80%, 90%, or 95% overlap with) the epitoperecognized by the antibody clone as described herein.

In some embodiments, an anti-TREM2 antibody recognizes an epitope ofhuman TREM2 that is the same or substantially the same as the epitoperecognized by an antibody clone selected from the group consisting ofClone CL0020306, Clone CL0020188, Clone CL0020307, and Clone CL0020123.

In some embodiments, an anti-TREM2 antibody binds to human TREM2 at anepitope within the stalk region of TREM2. In some embodiments, ananti-TREM2 antibody recognizes an epitope of human TREM2 comprising,within, or consisting of residues 129-172 or residues 131-169 of SEQ IDNO:1. In some embodiments, an anti-TREM2 antibody recognizes an epitopeof human TREM2 comprising, within, or consisting of residues 129-148 ofSEQ ID NO:1. In some embodiments, anti-TREM2 antibody recognizes anepitope of human TREM2 comprising, within, or consisting of amino acidresidues 143-149 of SEQ ID NO: 1. In some embodiments, an anti-TREM2antibody is an agonist that activates TREM2/DAP12 signaling (e.g., byinducing phosphorylation of a kinase such as Syk) and binds to humanTREM2 at an epitope within the stalk region of TREM2. In someembodiments, an anti-TREM2 antibody binds to human TREM2 at an epitopewithin the stalk region of TREM2 and inhibits cleavage of TREM2 by aprotease (e.g., ADAM17).

In some embodiments, an anti-TREM2 antibody binds to human TREM2 at anepitope within the Ig variable (IgV) domain of TREM2. In someembodiments, an anti-TREM2 antibody is an agonist that activatesTREM2/DAP12 signaling (e.g., by inducing phosphorylation of a kinasesuch as Syk) and binds to human TREM2 at an epitope within the IgVdomain of TREM2. In some embodiments, an anti-TREM2 antibody binds tohuman TREM2 at an epitope comprising or consisting of one or more of thefollowing: (i) amino acid residues 55-63 (GEKGPCQRV (SEQ ID NO:70)) ofSEQ ID NO:1, (ii) amino acids 96-107 (TLRNLQPHDAGL(SEQ ID NO:71)) of SEQID NO:1, and (iii) amino acid residues 126-129 (VEVL (SEQ ID NO:72)) ofSEQ ID NO:1.

Functional Characteristics of Anti-TREM2 Antibodies

In some embodiments, an anti-TREM2 antibody (e.g., an antibody havingone or more CDR, heavy chain variable region, and/or light chainvariable region sequences as disclosed) functions in one or more TREM2activities as disclosed herein. For example, in some embodiments ananti-TREM2 antibody is an antibody that modulates levels of sTREM2protein (e.g., levels of sTREM2 that are shed from the cell surface intoan extracellular sample), modulates recruitment or phosphorylation of akinase that interacts with a TREM2/DAP12 signaling complex (e.g., Sykkinase), and/or modulates one or more activities downstream of thesignaling complex, such as phagocytosis, cell growth, cell survival,cell differentiation, cytokine secretion, or cell migration. In someembodiments, an anti-TREM2 antibody as disclosed herein binds solubleTREM2 protein (sTREM2) in healthy human CSF or cynomolgus CSF withbetter potency compared to a reference antibody. In some embodiments,the reference antibody is represented by a combination of sequencesselected from the group consisting of: SEQ ID NOS:73 and 74; SEQ IDNOS:75 and 76; and SEQ ID NOS:77 and 78. In some embodiments, thepotency assay is carried out substantially as described in Example 11.

In some embodiments, an anti-TREM2 antibody enhances one or more TREM2activities (e.g., those described herein) that are induced by a ligand.In some embodiments, the ligand is a lipid ligand. Examples of TREM2lipid ligands include, but are not limited to,1-palmitoyl-2-(5′-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC),2-Arachidonoylglycerol (2-AG), 7-ketocholesterol (7-KC),24(S)hydroxycholesterol (240HC), 25(S)hydroxycholesterol (25OHC),27-hydroxycholesterol (270HC), Acyl Carnitine (AC),alkylacylglycerophosphocholine (PAF), α-galactosylceramide (KRN7000),Bis(monoacylglycero)phosphate (BMP), Cardiolipin (CL), Ceramide,Ceramide-1-phosphate (C1P), Cholesteryl ester (CE), Cholesterolphosphate (CP), Diacylglycerol 34:1 (DG 34:1), Diacylglycerol 38:4 (DG38:4), Diacylglycerol pyrophosphate (DGPP), Dihyrdoceramide (DhCer),Dihydrosphingomyelin (DhSM), Ether phosphatidylcholine (PCe), Freecholesterol (FC), Galactosylceramide (GalCer), Galactosyl sphingosine(GalSo), Ganglioside GM1, Ganglioside GM3, Glucosylsphingosine (GlcSo),Hank's Balanced Salt Solution (HBSS), Kdo2-Lipid A (KLA),Lactosylceramide (LacCer), lysoalkylacylglycerophosphocholine (LPAF),Lysophosphatidic acid (LPA), Lysophosphatidylcholine (LPC),Lysophosphatidylethanolamine (LPE), Lysophosphatidylglycerol (LPG),Lysophosphatidylinositol (LPI), Lysosphingomyelin (LSM),Lysophosphatidylserine (LPS), N-Acyl-phosphatidylethanolamine (NAPE),N-Acyl-Serine (NSer), Oxidized phosphatidylcholine (oxPC),Palmitic-acid-9-hydroxy-stearic-acid (PAHSA), Phosphatidylethanolamine(PE), Phosphatidylethanol (PEtOH), Phosphatidic acid (PA),Phosphatidylcholine (PC), Phosphatidylglycerol (PG),Phosphatidylinositol (PI), Phosphatidylserine (PS), Sphinganine,Sphinganine-1-phosphate (Sa1P), Sphingomyelin (SM), Sphingosine,Sphingosine-1-phosphate (So1P), and Sulfatide.

Modulation of sTREM2 Shedding

In some embodiments, an anti-TREM2 antibody alters levels of sTREM2protein in a sample, e.g., levels of sTREM2 that are shed from the cellsurface into an extracellular sample. In some embodiments, an anti-TREM2antibody decreases levels of sTREM2.

In some embodiments, an anti-TREM2 antibody decreases levels of sTREM2if the amount of sTREM2 in a treated sample is decreased by at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90% or more as compared to acontrol value. In some embodiments, an anti-TREM2 antibody decreaseslevels of sTREM2 if the amount of sTREM2 in a treated sample isdecreased by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold or more as compared to a control value. In someembodiments, the control value is the amount of sTREM2 in an untreatedsample (e.g., a supernatant from a TREM2-expressing cell that has notbeen treated with an anti-TREM2 antibody, or a sample from a subjectthat has not been treated with an anti-TREM2 antibody) or a sampletreated with an appropriate non-TREM2-binding antibody.

In some embodiments, sTREM2 shedding is measured using a sample thatcomprises a fluid, e.g., blood, plasma, serum, urine, or cerebrospinalfluid. In some embodiments, the sample comprises cerebrospinal fluid. Insome embodiments, the sample comprises supernatant from cell cultures(e.g., supernatant from a primary cell or cell line that endogenouslyexpresses TREM2, such as human macrophages, or a primary cell or cellline that has been engineered to express TREM2, e.g., as described inthe Examples section below).

In some embodiments, the level of sTREM2 in a sample is measured usingan immunoassay. Immunoassays are known in the art and include, but arenot limited to, enzyme immunoassays (EIA) such as enzyme multipliedimmunoassay (EMIA), enzyme-linked immunosorbent assay (ELISA),microparticle enzyme immunoassay (MEIA), immunohistochemistry (IHC),immunocytochemistry, capillary electrophoresis immunoassays (CEIA),radioimmunoassays (MA), immunofluorescence, chemiluminescenceimmunoassays (CL), and electrochemiluminescence immunoassays (ECL). Insome embodiments, sTREM2 levels are measuring using an ELISA assay. Insome embodiments, sTREM2 levels are measured using an ELISA assay asdescribed in the Examples section below.

Modulation of Kinase Recruitment or Phosphorylation

In some embodiments, an anti-TREM2 antibody induces phosphorylation of akinase that interacts with the TREM2/DAP12 signaling complex (such as,but not limited to, Syk, ZAP70, PI3K, Erk, AKT, or GSK3b). In someembodiments, an anti-TREM2 antibody induces phosphorylation of a kinasethat interacts with the TREM2/DAP12 signaling complex without blockingbinding of a native TREM2 ligand. In some embodiments, an anti-TREM2antibody enhances phosphorylation of a kinase that interacts with theTREM2/DAP12 signaling complex that is induced by a TREM2 ligand (e.g., alipid ligand). In some embodiments, an anti-TREM2 antibody induces orenhances phosphorylation of Syk. In some embodiments, an anti-TREM2antibody induces or enhances phosphorylation of Syk if the level of Sykphosphorylation in a sample treated with the anti-TREM2 antibody isincreased by at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90% ormore as compared to a control value. In some embodiments, an anti-TREM2antibody induces phosphorylation of Syk if the level of Sykphosphorylation in a sample treated with the anti-TREM2 antibody isincreased by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, or more as compared to a control value. In someembodiments, the control value is the level of Syk phosphorylation in anuntreated sample (e.g., a sample comprising a TREM2-expressing cell thathas not been treated with an anti-TREM2 antibody, or a sample from asubject that has not been treated with an anti-TREM2 antibody), or asample that has been treated with a TREM2 ligand but not an anti-TREM2antibody, or a sample treated with an appropriate non-TREM2-bindingantibody.

For detecting and/or quantifying phosphorylation (e.g., Sykphosphorylation) in a sample, in some embodiments, an immunoassay isused. In some embodiments, the immunoassay is an enzyme immunoassay(EIA), enzyme multiplied immunoassay (EMIA), enzyme-linked immunosorbentassay (ELISA), microparticle enzyme immunoassay (META),immunohistochemistry (IHC), immunocytochemistry, capillaryelectrophoresis immunoassay (CEIA), radioimmunoassay (MA),immunofluorescence, chemiluminescence immunoassay (CL), orelectrochemiluminescence immunoassay (ECL). In some embodiments,phosphorylation is detected and/or quantified using an immunoassay thatutilizes an amplified luminescent proximity homogenous assay(AlphaLISA®, PerkinElmer Inc.).

In some embodiments, phosphorylation is measured using a sample thatcomprises one or more cells, e.g., one or more TREM2-expressing cells(e.g., a primary cell or cell line that endogenously expresses TREM2,such as human macrophages or iPSC-derived microglia, or a primary cellor cell line that has been engineered to express TREM2, e.g., asdescribed in the Examples section below). In some embodiments, thesample comprises a fluid, e.g., blood, plasma, serum, urine, orcerebrospinal fluid. In some embodiments, the sample comprises tissue(e.g., lung, brain, kidney, spleen, nervous tissue, or skeletal muscle)or cells from such tissue. In some embodiments, the sample comprisesendogenous fluid, tissue, or cells (e.g., from a human or non-humansubject).

Modulation of Phagocytosis

In some embodiments, an anti-TREM2 antibody enhances phagocytosis ofdead cell debris, tissue debris, amyloid beta particles, or foreignmaterial. In some embodiments, an anti-TREM2 antibody enhancesphagocytosis without blocking binding of a native TREM2 ligand. In someembodiments, an anti-TREM2 antibody enhances phagocytosis that isinduced by a TREM2 ligand (e.g., a lipid ligand). In some embodiments,an anti-TREM2 antibody enhances phagocytosis if the level ofphagocytosis in a sample treated with the anti-TREM2 antibody isincreased by at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90% ormore as compared to a control value. In some embodiments, an anti-TREM2antibody enhances phagocytosis if the level of phagocytosis in a sampletreated with the anti-TREM2 antibody is increased by at least 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or moreas compared to a control value. In some embodiments, the control valueis the level of phagocytosis in an untreated sample, a sample that hasbeen treated with a TREM2 ligand but not an anti-TREM2 antibody, or asample treated with an appropriate non-TREM2-binding antibody.

In some embodiments, phagocytosis is measured using a phagocytosis assaywith a labeled substrate. Phagocytosis assays are known in the art. Insome embodiments, the phagocytosis assay is performed on a samplecomprising cells that endogenously express TREM2, such as humanmacrophages or microglia. In some embodiments, the phagocytosis assay isperformed on a sample comprising cells that have been engineered toexpress TREM2. In some embodiments, phagocytosis is measured using ahuman macrophage phagocytosis assay as described in the Examples sectionbelow.

Modulation of Cell Differentiation, Function, Migration, and Survival

In some embodiments, an anti-TREM2 antibody enhances cell migration,cell survival, cell function, or cell differentiation (e.g., for myeloidcells, macrophages, and microglia, including iPSC-derived microglia anddisease-associated microglia). Disease-associated microglia and methodsof detecting disease-associated microglia are described in Keren-Shaulet al., Cell, 2017, 169:1276-1290. In some embodiments, an anti-TREM2antibody enhances cell migration of one or more cell types (e.g.,myeloid cells, macrophages, or microglia). In some embodiments, ananti-TREM2 antibody enhances cell survival of one or more cell types(e.g., myeloid cells, macrophages, or microglia). In some embodiments,an anti-TREM2 antibody enhances cell function of one or more cell types(e.g., myeloid cells, macrophages, or microglia). In some embodiments,an anti-TREM2 antibody enhances cell differentiation of one or more celltypes (e.g., myeloid cells, macrophages, or microglia). In someembodiments, an anti-TREM2 antibody enhances the migration, survival,function, and/or differentiation of myeloid cells. In some embodiments,an anti-TREM2 antibody enhances the migration, survival, function,and/or differentiation of macrophages. In some embodiments, ananti-TREM2 antibody enhances the migration, survival, function, and/ordifferentiation of microglia. In some embodiments, an anti-TREM2antibody enhances microglia activation. In some embodiments, ananti-TREM2 antibody enhances the migration, survival, function, and/ordifferentiation of disease-associated microglia. In some embodiments, ananti-TREM2 antibody enhances cell migration, cell survival, cellfunction, or cell differentiation without blocking binding of a nativeTREM2 ligand. In some embodiments, an anti-TREM2 antibody enhances cellmigration, cell survival, cell function, or cell differentiation that isinduced by a TREM2 ligand (e.g., a lipid ligand).

In some embodiments, an anti-TREM2 antibody enhances cell migration,cell survival, cell function, or cell differentiation if the level ofactivity (e.g., migration, survival, function, or differentiation) in asample treated with the anti-TREM2 antibody is increased by at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90% or more as compared to acontrol value. In some embodiments, an anti-TREM2 antibody enhances cellmigration, cell survival, cell function, or cell differentiation if thelevel of activity (e.g., migration, survival, function, ordifferentiation) in a sample treated with the anti-TREM2 antibody isincreased by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, or more as compared to a control value. In someembodiments, the control value is the level of activity (e.g.,migration, survival, function, or differentiation) in an untreatedsample (e.g., a sample that has not been treated with an anti-TREM2antibody), a sample that has been treated with a TREM2 ligand but not ananti-TREM2 antibody, or a sample treated with an appropriatenon-TREM2-binding antibody.

In some embodiments, cell migration is measured using a chemotaxisassay. Chemotaxis assays are known in the art. In some embodiments, thecell migration assay (e.g., chemotaxis assay) is performed on a samplecomprising cells that endogenously express TREM2, such as humanmacrophages. In some embodiments, the cell migration assay (e.g.,chemotaxis assay) is performed on a sample comprising cells that havebeen engineered to express TREM2. In some embodiments, cell migration ismeasured using a human macrophage chemotaxis assay as described in theExamples section below.

In some embodiments, cell survival is measured using a cell viabilityassay. Cell viability assays are known in the art. In some embodiments,the cell survival assay (e.g., cell viability assay) is performed on asample comprising cells that endogenously express TREM2, such as humanmacrophages. In some embodiments, the cell survival assay (e.g., cellviability assay) is performed on a sample comprising cells that havebeen engineered to express TREM2. In some embodiments, cell survival ismeasured using a human macrophage viability assay as described in theExamples section below.

In some embodiments, cell function is measured using a functional assaythat is appropriate for that cell. For example, in some embodiments,macrophage cell function is evaluated using a phagocytosis assay, e.g.,as described in the Examples section below.

In some embodiments, cell differentiation is measured by evaluating theability of cells that endogenously express TREM2 to differentiate. Forexample, in some embodiments, cell differentiation is measured byevaluating the ability of macrophages to differentiate from monocytes,e.g., as described in the Examples section below.

In some embodiments, activation of microglia is measured in vivo. Insome embodiments, microglia activation is measured using TSPO-PETimaging. TSPO-PET imaging methods are known in the art.

In some embodiments, an anti-TREM2 antibody enhances microglia functionwithout increasing neuroinflammation. Levels of neuroinflammation can bedetermined by measuring levels of cytokines (e.g., inflammatorycytokines), such as but not limited to TNF-α, IL-1β, IL-6, IL-1ra, TGFβ,IL-15, or IFN-γ. In some embodiments, cytokine levels are measured usingimmunoassays, for example, an enzyme immunoassay (EIA), enzymemultiplied immunoassay (EMIA), enzyme-linked immunosorbent assay(ELISA), microparticle enzyme immunoassay (MEIA), immunohistochemistry(IHC), immunocytochemistry, capillary electrophoresis immunoassay(CEIA), radioimmunoassay (MA), immunofluorescence, chemiluminescenceimmunoassay (CL), or electrochemiluminescence immunoassay (ECL).

IV. Preparation of Antibodies

In some embodiments, antibodies are prepared by immunizing an animal oranimals (e.g., mice, rabbits, or rats) with an antigen or a mixture ofantigens for the induction of an antibody response. In some embodiments,the antigen or mixture of antigens is administered in conjugation withan adjuvant (e.g., Freund's adjuvant). After an initial immunization,one or more subsequent booster injections of the antigen or antigens maybe administered to improve antibody production. Following immunization,antigen-specific B cells are harvested, e.g., from the spleen and/orlymphoid tissue. For generating monoclonal antibodies, the B cells arefused with myeloma cells, which are subsequently screened for antigenspecificity. Methods of preparing antibodies are also described in theExamples section below.

The genes encoding the heavy and light chains of an antibody of interestcan be cloned from a cell, e.g., the genes encoding a monoclonalantibody can be cloned from a hybridoma and used to produce arecombinant monoclonal antibody. Gene libraries encoding heavy and lightchains of monoclonal antibodies can also be made from hybridoma orplasma cells. Alternatively, phage or yeast display technology can beused to identify antibodies and Fab fragments that specifically bind toselected antigens. Antibodies can also be made bispecific, i.e., able torecognize two different antigens. Antibodies can also beheteroconjugates, e.g., two covalently joined antibodies, orimmunotoxins.

Antibodies can be produced using any number of expression systems,including prokaryotic and eukaryotic expression systems. In someembodiments, the expression system is a mammalian cell expression, suchas a hybridoma, or a CHO cell expression system. Many such systems arewidely available from commercial suppliers. In embodiments in which anantibody comprises both a V_(H) and V_(L) region, the V_(H) and V_(L)regions may be expressed using a single vector, e.g., in a di-cistronicexpression unit, or under the control of different promoters. In otherembodiments, the V_(H) and V_(L) region may be expressed using separatevectors. A V_(H) or V_(L) region as described herein may optionallycomprise a methionine at the N-terminus.

In some embodiments, the antibody is a chimeric antibody. Methods formaking chimeric antibodies are known in the art. For example, chimericantibodies can be made in which the antigen binding region (heavy chainvariable region and light chain variable region) from one species, suchas a mouse, is fused to the effector region (constant domain) of anotherspecies, such as a human. As another example, “class switched” chimericantibodies can be made in which the effector region of an antibody issubstituted with an effector region of a different immunoglobulin classor subclass.

In some embodiments, the antibody is a humanized antibody. Generally, anon-human antibody is humanized in order to reduce its immunogenicity.Humanized antibodies typically comprise one or more variable regions(e.g., CDRs) or portions thereof that are non-human (e.g., derived froma mouse variable region sequence), and possibly some framework regionsor portions thereof that are non-human, and further comprise one or moreconstant regions that are derived from human antibody sequences. Methodsfor humanizing non-human antibodies are known in the art. Transgenicmice, or other organisms such as other mammals, can be used to expresshumanized or human antibodies. Other methods of humanizing antibodiesinclude, for example, variable domain resurfacing, CDR grafting,grafting specificity-determining residues (SDR), guided selection, andframework shuffling.

As an alternative to humanization, fully human antibodies can begenerated. As a non-limiting example, transgenic animals (e.g., mice)can be produced that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (JH) genein chimeric and germ-line mutant mice results in complete inhibition ofendogenous antibody production. Transfer of the human germ-lineimmunoglobulin gene array in such germ-line mutant mice will result inthe production of human antibodies upon antigen challenge. As anotherexample, human antibodies can be produced by hybridoma-based methods,such as by using primary human B cells for generating cell linesproducing human monoclonal antibodies.

Human antibodies can also be produced using phage display or yeastdisplay technology. In phage display, repertoires of variable heavychain and variable light chain genes are amplified and expressed inphage display vectors. In some embodiments, the antibody library is anatural repertoire amplified from a human source. In some embodiments,the antibody library is a synthetic library made by cloning heavy chainand light chain sequences and recombining to generate a large pool ofantibodies with different antigenic specificity. Phage typically displayantibody fragments (e.g., Fab fragments or scFv fragments), which arethen screened for binding to an antigen of interest.

In some embodiments, antibody fragments (such as a Fab, a Fab′, aF(ab′)₂, a scFv, a V_(H), or a V_(HH)) are generated. Various techniqueshave been developed for the production of antibody fragments.Traditionally, these fragments were derived via proteolytic digestion ofintact antibodies. However, these fragments can now be produced directlyusing recombinant host cells. For example, antibody fragments can beisolated from antibody phage libraries. Alternatively, Fab′-SH fragmentscan be directly recovered from E. coli cells and chemically coupled toform F(ab′)2 fragments. According to another approach, F(ab′)2 fragmentscan be isolated directly from recombinant host cell culture. Othertechniques for the production of antibody fragments will be apparent tothose skilled in the art.

In some embodiments, an antibody or an antibody fragment is conjugatedto another molecule, e.g., polyethylene glycol (PEGylation) or serumalbumin, to provide an extended half-life in vivo.

In some embodiments, multispecific antibodies comprising an anti-TREM2antibody (or antigen-binding fragment thereof) as described herein areprovided, e.g., a bispecific antibody. Multispecific antibodies areantibodies that have binding specificities for at least two differentsites. In some embodiments, a multispecific antibody (e.g., a bispecificantibody) has a binding specificity for TREM2 and has a bindingspecificity for at least one other antigen. In some embodiments, amultispecific antibody (e.g., a bispecific antibody) binds to twodifferent TREM2 epitopes. In some embodiments, a multispecific antibody(e.g., a bispecific antibody) is capable of inducing TREM2 clustering atthe cell surface. An illustrative method for measuring receptorclustering using confocal FRET microscopy is described in Wallrabe etal., Biophys. 1, 2003, 85:559-571. Methods of making multispecificantibodies (e.g., bispecific antibodies) include, but are not limitedto, recombinant co-expression of two pairs of heavy chain and lightchain in a host cell, “knobs-into-holes” engineering, intramoleculartrimerization, and fusion of an antibody fragment to the N-terminus orC-terminus of another antibody, e.g., tandem variable domains.

V. Nucleic Acids, Vectors, and Host Cells

In some embodiments, the anti-TREM2 antibodies as disclosed herein areprepared using recombinant methods. Accordingly, in some aspects, thedisclosure provides isolated nucleic acids comprising a nucleic acidsequence encoding any of the anti-TREM2 antibodies as described herein(e.g., any one or more of the CDRs, heavy chain variable regions, andlight chain variable regions described herein); vectors comprising suchnucleic acids; and host cells into which the nucleic acids areintroduced that are used to replicate the antibody-encoding nucleicacids and/or to express the antibodies.

In some embodiments, a polynucleotide (e.g., an isolated polynucleotide)comprises a nucleotide sequence encoding an antibody or antigen-bindingportion thereof as described herein (e.g., as described in the Sectionabove entitled “Anti-TREM2 Antibody Sequences”). In some embodiments,the polynucleotide comprises a nucleotide sequence encoding one or moreamino acid sequences (e.g., CDR, heavy chain, or light chain sequences)disclosed in the Informal Sequence Listing below. In some embodiments,the polynucleotide comprises a nucleotide sequence encoding an aminoacid sequence having at least 85% sequence identity (e.g., at least 85%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity) to a sequence (e.g., a CDR, heavy chain, or lightchain sequence) disclosed in the Informal Sequence Listing below. Insome embodiments, a polynucleotide as described herein is operablylinked to a heterologous nucleic acid, e.g., a heterologous promoter.

Suitable vectors containing polynucleotides encoding antibodies of thepresent disclosure, or fragments thereof, include cloning vectors andexpression vectors. While the cloning vector selected may vary accordingto the host cell intended to be used, useful cloning vectors generallyhave the ability to self-replicate, may possess a single target for aparticular restriction endonuclease, and/or may carry genes for a markerthat can be used in selecting clones containing the vector. Examplesinclude plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript(e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColEl,pCR1, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28. Theseand many other cloning vectors are available from commercial vendorssuch as BioRad, Strategene, and Invitrogen.

Expression vectors generally are replicable polynucleotide constructsthat contain a nucleic acid of the present disclosure. The expressionvector may replicate in the host cells either as episomes or as anintegral part of the chromosomal DNA. Suitable expression vectorsinclude but are not limited to plasmids, viral vectors, includingadenoviruses, adeno-associated viruses, retroviruses, and any othervector.

Suitable host cells for cloning or expressing a polynucleotide or vectoras described herein include prokaryotic or eukaryotic cells. In someembodiments, the host cell is prokaryotic. In some embodiments, the hostcell is eukaryotic, e.g., Chinese Hamster Ovary (CHO) cells or lymphoidcells. In some embodiments, the host cell is a human cell, e.g., a HumanEmbryonic Kidney (HEK) cell.

In another aspect, methods of making an anti-TREM2 antibody as describedherein are provided. In some embodiments, the method includes culturinga host cell as described herein (e.g., a host cell expressing apolynucleotide or vector as described herein) under conditions suitablefor expression of the antibody. In some embodiments, the antibody issubsequently recovered from the host cell (or host cell culture medium).

VI. Therapeutic Methods Using Anti-Trem2 Antibodies

In another aspect, therapeutic methods using an anti-TREM2 antibody asdisclosed herein (e.g., an anti-TREM2 antibody as described in SectionIII above) are provided. In some embodiments, methods of treating aneurodegenerative disease are provided. In some embodiments, methods ofmodulating one or more TREM2 activities (e.g., in a subject having aneurodegenerative disease) are provided.

In some embodiments, methods of treating a neurodegenerative disease areprovided. In some embodiments, the neurodegenerative disease is selectedfrom the group consisting of Alzheimer's disease, primary age-relatedtauopathy, progressive supranuclear palsy (PSP), frontotemporaldementia, frontotemporal dementia with parkinsonism linked to chromosome17, argyrophilic grain dementia, amyotrophic lateral sclerosis,amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam(ALS-PDC), corticobasal degeneration, chronic traumatic encephalopathy,Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillarytangles with calcification, Down's syndrome, familial British dementia,familial Danish dementia, Gerstmann-Straussler-Scheinker disease,globular glial tauopathy, Guadeloupean parkinsonism with dementia,Guadelopean PSP, Hallevorden-Spatz disease, hereditary diffuseleukoencephalopathy with spheroids (HDLS), Huntington's disease,inclusion-body myositis, multiple system atrophy, myotonic dystrophy,Nasu-Hakola disease, neurofibrillary tangle-predominant dementia,Niemann-Pick disease type C, pallido-ponto-nigral degeneration,Parkinson's disease, Pick's disease, postencephalitic parkinsonism,prion protein cerebral amyloid angiopathy, progressive subcorticalgliosis, subacute sclerosing panencephalitis, and tangle only dementia.In some embodiments, the neurodegenerative disease is Alzheimer'sdisease. In some embodiments, the neurodegenerative disease isNasu-Hakola disease. In some embodiments, the neurodegenerative diseaseis frontotemporal dementia. In some embodiments, the neurodegenerativedisease is Parkinson's disease. In some embodiments, the methodcomprises administering to the subject an isolated antibody or anantigen-binding fragment thereof that specifically binds to a humanTREM2 protein, e.g., an anti-TREM2 antibody as described herein, or apharmaceutical composition comprising an anti-TREM2 antibody asdescribed herein.

In some embodiments, an anti-TREM2 antibody (or antigen-binding portionor pharmaceutical composition thereof) as described herein is used intreating a neurodegenerative disease that is characterized by a mutationin TREM2. In some embodiments, the neurodegenerative disease that ischaracterized by a mutation in TREM2 is Alzheimer's disease, e.g.,Alzheimer's disease that is characterized by a R47H mutation in TREM2.

In some embodiments, methods of modulating one or more TREM2 activitiesin a subject (e.g., a subject having a neurodegenerative disease) areprovided. In some embodiments, the method comprises modulating levels ofsTREM2; modulating recruitment or phosphorylation of a kinase thatinteracts with a TREM2/DAP12 signaling complex (e.g., Syk kinase);modulating phagocytosis (e.g., phagocytosis of cell debris, amyloid betaparticles, etc.); modulating cell migration (e.g., migration of myeloidcells, macrophages, microglia, and disease associated microglia); and/ormodulating cell differentiation (e.g., for myeloid cells, macrophages,microglia, and disease associated microglia). In some embodiments,methods of enhancing one or more TREM2 activities in a subject having aneurodegenerative disease are provided. In some embodiments, methods ofdecreasing levels of sTREM2 in a subject having a neurodegenerativedisease are provided. In some embodiments, the method of modulating oneor more TREM2 activities in a subject comprises administering to thesubject an isolated antibody or an antigen-binding portion thereof thatspecifically binds to a human TREM2 protein, e.g., an anti-TREM2antibody as describe herein, or a pharmaceutical composition comprisingan anti-TREM2 antibody as described herein.

In some embodiments, the subject to be treated is a human, e.g., a humanadult or a human child.

In some embodiments, methods of reducing plaque accumulation in asubject having a neurodegenerative disease are provided. In someembodiments, the method comprises administering to the subject anantibody or pharmaceutical composition as described herein. In someembodiments, the subject has Alzheimer's disease. In some embodiments,the subject is an animal model of a neurodegenerative disease (e.g., a5×FAD or APP/PS1 mouse model). In some embodiments, plaque accumulationis measured by amyloid plaque imaging and/or Tau imaging, e.g., usingpositron emission tomography (PET) scanning. In some embodiments,administration of an anti-TREM2 antibody reduces plaque accumulation byat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, or at least 90% as compared to a baseline value(e.g., the level of plaque accumulation in the subject prior toadministration of the anti-TREM2 antibody).

In some embodiments, an anti-TREM2 antibody is administered to a subjectat a therapeutically effective amount or dose. A daily dose range ofabout 0.01 mg/kg to about 500 mg/kg, or about 0.1 mg/kg to about 200mg/kg, or about 1 mg/kg to about 100 mg/kg, or about 10 mg/kg to about50 mg/kg, can be used. The dosages, however, may be varied according toseveral factors, including the chosen route of administration, theformulation of the composition, patient response, the severity of thecondition, the subject's weight, and the judgment of the prescribingphysician. The dosage can be increased or decreased over time, asrequired by an individual patient. In certain instances, a patientinitially is given a low dose, which is then increased to an efficaciousdosage tolerable to the patient. Determination of an effective amount iswell within the capability of those skilled in the art.

The route of administration of an anti-TREM2 antibody as describedherein can be oral, intraperitoneal, transdermal, subcutaneous,intravenous, intramuscular, intrathecal, inhalational, topical,intralesional, rectal, intrabronchial, nasal, transmucosal, intestinal,ocular or otic delivery, or any other methods known in the art. In someembodiments, the antibody is administered orally, intravenously, orintraperitoneally.

In some embodiments, the anti-TREM2 antibody (and optionally anothertherapeutic agent) is administered to the subject over an extendedperiod of time, e.g., for at least 30, 40, 50, 60, 70, 80, 90, 100, 150,200, 250, 300, 350 days or longer.

VII. Pharmaceutical Compositions and Kits

In another aspect, pharmaceutical compositions and kits comprising anantibody that specifically binds to a human TREM2 protein are provided.In some embodiments, the pharmaceutical compositions and kits are foruse in treating a neurodegenerative disease. In some embodiments, thepharmaceutical compositions and kits are for use in modulating (e.g.,enhancing or inhibiting) one or more TREM2 activities, e.g., Sykphosphorylation. In some embodiments, the pharmaceutical compositionsand kits are for use in modulating (e.g., decreasing) sTREM2 levels.

Pharmaceutical Compositions

In some embodiments, pharmaceutical compositions comprising ananti-TREM2 antibody or an antigen-binding fragment thereof are provided.In some embodiments, the anti-TREM2 antibody is an antibody as describedin Section III above or an antigen-binding fragment thereof.

In some embodiments, a pharmaceutical composition comprises ananti-TREM2 antibody as described herein and further comprises one ormore pharmaceutically acceptable carriers and/or excipients. Apharmaceutically acceptable carrier includes any solvents, dispersionmedia, or coatings that are physiologically compatible and that does notinterfere with or otherwise inhibit the activity of the active agent.Various pharmaceutically acceptable excipients are well-known in theart.

In some embodiments, the carrier is suitable for intravenous,intramuscular, oral, intraperitoneal, intrathecal, transdermal, topical,or subcutaneous administration. Pharmaceutically acceptable carriers cancontain one or more physiologically acceptable compound(s) that act, forexample, to stabilize the composition or to increase or decrease theabsorption of the active agent(s). Physiologically acceptable compoundscan include, for example, carbohydrates, such as glucose, sucrose, ordextrans, antioxidants, such as ascorbic acid or glutathione, chelatingagents, low molecular weight proteins, compositions that reduce theclearance or hydrolysis of the active agents, or excipients or otherstabilizers and/or buffers. Other pharmaceutically acceptable carriersand their formulations are well-known in the art.

The pharmaceutical compositions described herein can be manufactured ina manner that is known to those of skill in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making,emulsifying, encapsulating, entrapping or lyophilizing processes. Thefollowing methods and excipients are merely exemplary and are in no waylimiting.

For oral administration, an anti-TREM2 antibody can be formulated bycombining it with pharmaceutically acceptable carriers that are wellknown in the art. Such carriers enable the compounds to be formulated astablets, pills, dragees, capsules, emulsions, lipophilic and hydrophilicsuspensions, liquids, gels, syrups, slurries, suspensions and the like,for oral ingestion by a patient to be treated. Pharmaceuticalpreparations for oral use can be obtained by mixing the compounds with asolid excipient, optionally grinding a resulting mixture, and processingthe mixture of granules, after adding suitable auxiliaries, if desired,to obtain tablets or dragee cores. Suitable excipients include, forexample, fillers such as sugars, including lactose, sucrose, mannitol,or sorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents can be added, such as a cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate.

An anti-TREM2 antibody can be formulated for parenteral administrationby injection, e.g., by bolus injection or continuous infusion. Forinjection, the compound or compounds can be formulated into preparationsby dissolving, suspending or emulsifying them in an aqueous ornonaqueous solvent, such as vegetable or other similar oils, syntheticaliphatic acid glycerides, esters of higher aliphatic acids or propyleneglycol; and if desired, with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives. In some embodiments, compounds can beformulated in aqueous solutions, e.g., in physiologically compatiblebuffers such as Hanks's solution, Ringer's solution, or physiologicalsaline buffer. Formulations for injection can be presented in unitdosage form, e.g., in ampules or in multi-dose containers, with an addedpreservative. The compositions can take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and can containformulatory agents such as suspending, stabilizing and/or dispersingagents.

In some embodiments, an anti-TREM2 antibody is prepared for delivery ina sustained-release, controlled release, extended-release, timed-releaseor delayed-release formulation, for example, in semi-permeable matricesof solid hydrophobic polymers containing the active agent. Various typesof sustained-release materials have been established and are well knownby those skilled in the art. Current extended-release formulationsinclude film-coated tablets, multiparticulate or pellet systems, matrixtechnologies using hydrophilic or lipophilic materials and wax-basedtablets with pore-forming excipients. Sustained-release delivery systemscan, depending on their design, release the compounds over the course ofhours or days, for instance, over 4, 6, 8, 10, 12, 16, 20, 24 hours ormore. Usually, sustained release formulations can be prepared usingnaturally-occurring or synthetic polymers, for instance, polymeric vinylpyrrolidones, such as polyvinyl pyrrolidone (PVP); carboxyvinylhydrophilic polymers; hydrophobic and/or hydrophilic hydrocolloids, suchas methylcellulose, ethylcellulose, hydroxypropylcellulose, andhydroxypropylmethylcellulose; and carboxypolymethylene.

Typically, a pharmaceutical composition for use in in vivoadministration is sterile. Sterilization can be accomplished accordingto methods known in the art, e.g., heat sterilization, steamsterilization, sterile filtration, or irradiation.

Dosages and desired drug concentration of pharmaceutical compositions ofthe disclosure may vary depending on the particular use envisioned. Thedetermination of the appropriate dosage or route of administration iswell within the skill of one in the art. Suitable dosages are alsodescribed in Section VI above.

Kits

In some embodiments, kits comprising an anti-TREM2 antibody or anantigen-binding fragment thereof are provided. In some embodiments, theanti-TREM2 antibody is an antibody as described in Section III above oran antigen-binding fragment thereof.

In some embodiments, the kit further comprises one or more additionaltherapeutic agents. For example, in some embodiments, the kit comprisesan anti-TREM2 antibody as described herein and further comprises one ormore additional therapeutic agents for use in the treatment of aneurodegenerative disease, e.g., Alzheimer's disease. In someembodiments, the therapeutic agent is an agent for use in treating acognitive or behavioral symptom of a neurodegenerative disease (e.g., anantidepressant, a dopamine agonist, or an anti-psychotic). In someembodiments, the therapeutic agent is a neuroprotective agent (e.g.,carbidopa/levodopa, an anticholinergic agent, a dopaminergic agent, amonoamine oxidase B (MAO-B) inhibitor, a catechol-O-methyl transferase(COMT) inhibitor, a glutamatergic agent, a histone deacetylase (HDAC)inhibitor, a cannabinoid, a caspase inhibitor, melatonin, ananti-inflammatory agent, a hormone (e.g., estrogen or progesterone), ora vitamin).

In some embodiments, the kit comprises an anti-TREM antibody asdescribed herein and further comprises one or more reagents formeasuring sTREM2 levels. In some embodiments, the kit comprises ananti-TREM antibody as described herein and further comprises one or morereagents for measuring TREM2 activity (e.g., for measuring Sykphosphorylation).

In some embodiments, the kit further comprises instructional materialscontaining directions (i.e., protocols) for the practice of the methodsdescribed herein (e.g., instructions for using the kit for a therapeuticmethod as described in Section VI above). While the instructionalmaterials typically comprise written or printed materials, they are notlimited to such. Any medium capable of storing such instructions andcommunicating them to an end user is contemplated by this disclosure.Such media include, but are not limited to electronic storage media(e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g.,CD-ROM), and the like. Such media may include addresses to internetsites that provide such instructional materials.

VIII. Examples

The present invention will be described in greater detail by way ofspecific examples. The following examples are offered for illustrativepurposes only, and are not intended to limit the invention in anymanner.

Example 1. Generation and Initial Characterization of Anti-TREM2Antibodies Recombinant Expression and Purification of Mouse Fc FusedHuman TREM2 ECD

The ecto domain (residues 19-172) of human TREM2 (UniProtKB ID—Q9NZC2)was subcloned into pRK vector with the secretion signal from mouse IgGkappa chain V-III, amino acids 1-20 (UniProtKB ID-P01661) at theN-terminal region, and a mouse Fc tag at the C-terminal region with aGGGGS (SEQ ID NO:64) between TREM2 ECD and Fc.

Purified plasmid was transfected into Expi293F™ cells (Thermo Fisher)using the Expi293F™ Expression System Kit according to themanufacturer's instructions. To inhibit maturation of N-linked glycansand reduce glycosylation heterogeneity, kifunensine (Sigma), aninhibitor of high mannosidase I was added to the culture at 1 μg/mLconcentration immediately after transfection. Transfected cells wereincubated in an orbital shaker (Infors HT Multitron) at 125 rpm and 37°C. in a humidified atmosphere of 6% CO₂. ExpiFectamine™ 293 TransfectionEnhancer 1 and 2 were added to the cells 16 hours post transfection andthe media supernatant was harvested 96 hours post transfection. Theclarified supernatant was supplemented with EDTA-free protease inhibitor(Roche) and was stored at −80° C.

For rhTREM2-Fc isolation, clarified media supernatant was loaded onHiTrap Mab Select SuRe Protein A affinity column (GE Healthcare LifeSciences) and washed with 200 mM arginine and 137 mM succinate buffer pH5.0. The fusion protein was eluted in 100 mM QB citrate buffer pH 3.0and 50 mM NaCl. Immediately after elution, 1M Tris-HCl buffer pH 8.0 wasadded to the protein solution to neutralize the pH. Protein aggregateswere separated by size exclusion chromatography (SEC) on Superdex 200increase 10/300 GL column (GE Healthcare Life Sciences). The SEC mobilephase buffer was kept at 20 mM Tris-HCl pH 8.0, 100 mM NaCl and 50 mMarginine, which was also the protein storage buffer. All chromatographysteps were performed on AKTA pure or AKTA Avant systems (GE HealthcareLife Sciences).

Recombinant Expression and Purification of His-Tagged TREM2 ECD

The ecto domain (residues 19-172) of TREM2 (UniProtKB—Q9NZC2) wassubcloned in the pRK vector with the secretion signal from mouse Igkappa chain V-III, amino acids 1-20 (UniProtKB ID—P01661) at theN-terminal region, and a 6×-His tag (SEQ ID NO:65) at the C-terminalregion. The insert was verified by sequencing and maxi prep plasmidpurification was performed.

Purified plasmid was transfected into Expi293F™ cells (Thermo Fisher)using the Expi293F™ Expression System Kit according to themanufacturer's instructions. Transfected cells were incubated in anorbital shaker (Infors HT Multitron) at 125 rpm and 37° C. in ahumidified atmosphere of 6% CO2. ExpiFectamine™ 293 TransfectionEnhancer 1 and 2 were added to the cells 16 hours post transfection andthe media supernatant was harvested 96 hours post transfection.

Harvested media was supplemented with 1M imidazole pH 8.0 to a finalconcentration of 10 mM and filtered using the Nalgene™ Rapid-Flow™disposable filter units (Thermo Fisher) with a pore size of 0.4 microns.HisPur™ Ni-NTA Resin (Thermo Fisher) was washed with MQ water andequilibrated with load buffer (20 mM Tris pH 8.0, 150 mM NaCl, and 10 mMimidazole). Affinity purification was performed using the gravity flowmethod. The harvested media was loaded onto the resin andnonspecifically bound proteins were washed with load buffer supplementedwith 50 mM and 100 mM imidazole. The bound His-tagged TREM2 eco domainwas eluted with 20 mM Tris pH 8.0, 150 mM NaCl, and 200 mM imidazole.Eluted protein was concentrated using Amicon 10 kDa concentrators andthe concentrated protein was further purified by gel filtrationchromatography using the AKTA Avant system (GE Healthcare LifeSciences). The protein was loaded onto a HiLoad Superdex 200 16/600 (GEHealthcare Life Sciences) column equilibrated with 1×PBS and eluted andfractionated using 1×PBS as the running buffer. Eluted fractions wereanalyzed by electrophoresis on polyacrylamide (PAGE) gels underdenaturing and native conditions. Eluted fractions were furthercharacterized by analytical size exclusion chromatography and the intactprotein mass determination. Results from the PAGE and analyticalcharacterization were used to pool the heavily glycosylated proteinfractions and these were aliquoted and stored at −80° C.

Generation of Antibodies

Rodents (mice and rats) were immunized using standard protocols withrhTREM2-Fc immunogen or BWZ cells expressing full length Trem2 receptor.Titers were measured throughout immunization using sera collected atdifferent time points. The detection of an antigen specific immuneresponse was performed using flow cytometry with the rhTREM2-Fcimmunogen and live BWZ cells expressing full-length TREM2. Selectioncriteria of candidate antibodies included rodent antibody production andspecificity of binding to TREM2 as detected by flow cytometry.Antibody-secreting cells were isolated from animal immune tissuesincluding spleen, lymph nodes and bone marrow.

Single cell suspensions were analyzed to determine the bindingproperties of secreted antibodies. Antibody-secreting cells were loadedinto microfluidic devices and isolated in nanoliter volume reactionchambers to enable the detection of secreted antibodies usingfluorescent and brightfield image-based microscopy assays (see, e.g.,U.S. Pat. No. 9,188,593). Binding assays involving detection ofantibodies binding to antigen-coated micro-beads, detection of solublefluorescently-labeled antigen binding to antibodies immobilized onbeads, and detection of antibody binding to cell surface-expressedantigens were carried out. Cell surface-expressed antigens included bothrecombinant form and the native forms of antigens presented on thesurface of cells.

Image analysis was used to identify chambers exhibiting positivefluorescent signals, indicating the presence of a single cell producingantibodies with the desired properties, and the contents of chamberswere recovered and lysed in 384 well plates (see, e.g., U.S. Pat. No.10,087,408). Single cell lysates were then subjected to RT-PCR toamplify the heavy and light chain variable region sequences. Theresulting amplicons were then sequenced to determine the cDNA sequenceof paired heavy and light chain variable regions from the selectedsingle cells. The resulting sequences were manually inspected andanalyzed to determine sequence diversity and somatic hypermutation.Sequences were selected for expression based on screening data andsequence diversity. Expressed antibodies were tested to confirm antigenbinding specificity.

Primary Screening of Anti-TREM2 Antibodies

Primary screening of antibodies was performed in HEK 293 cellsexpressing TREM2, wild-type iPSC, and TREM2 knockout iPSC as follows.

1. Screening for TREM2 Binding in TREM2-Expressing HEK Cells

A HEK 293 cell line stably expressing human TREM2/DAP12 was generated bytransfecting the cells with a vector expressing wild type human TREM2and DAP12, and DAP12 alone, respectively. Stable expressing clones wereselected, and the cell surface TREM2 expression was evaluated by flowcytometry. APC-conjugated rat anti-human/mouse-TREM2 monoclonal antibody(R&D, Catalog No. MAB17291) was used to detect surface TREM2 expression.The clone showing the highest wild type TREM2 expression level wasselected and named “HEK293-H6.” The clones stably expressing DAP12 wereanalyzed by Western blot, and the selected clone was named “HEK293-DAP12#1.”

HEK 293 overexpressing human TREM2 (HEK293-H6) and HEK 293overexpressing GFP (B5) were harvested by 0.05% trypsin and incubated at37° C. for 2 hours. After incubation, the cells were centrifuged andwashed in FACS buffer (PBS+0.5% BSA) twice. Mixed cells were resuspendedin FACS buffer with human Trustain FcX solution (Biolegend, Catalog No.422302) at a density of 10⁶/mL per cell line. The mixed cell lines wereseeded at 200,000 cells per well in a 96-well round-bottom plate andincubated for 20 minutes at room temperature. After incubation, thecells were centrifuged and incubated with anti-TREM2 antibodies of about0-200 nM dose titration for 45 minutes on ice. After incubation, thecells were centrifuged and washed with FACS buffer three times. Thecells were then incubated with secondary antibody (Alexa Fluor 647AffiniPure F(ab′)2 Fragment Goat Anti-human IgG(H+L), JacksonImmunoResearch Laboratories, Catalog No. 109-606-088, 1:800 dilution)for 30 minutes on ice. After incubation, the cells were washed with FACSbuffer three times, resuspended in 100 μL of FACS buffer, and analyzedby flow cytometry (BD FACSCanto II, San Jose, Calif.), for which 30,000events were obtained for each sample. Mean fluorescence intensity percells were calculated by FlowJo software and used for generating doseresponse binding curve.

FIG. 1 illustrates a representative result for an exemplary antibodythat binds cell surface receptor TREM2 in HEK293-H6 cells.

Evaluation of Activation of TREM2-Dependent pSyk Signaling

Activation of TREM2-dependent pSyk signaling was measured in humanmacrophage cells or in HEK293-H6 cells using a commercial AlphaLisaassay from Perkin-Elmer.

For all experiments involving use of lipid vesicles containing 70% DOPCand 30% POPS, the lipid vesicles were prepared within two weeks ofexperiments as follows: 7 mg DOPC(1,2-dioleoyl-sn-glycero-3-phosphocholine) and 3 mg POPS(1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine) were combined inchloroform in a glass vial and dried under a stream of N2 gas for 1-2hours, or until completely dry. The lipid mixture was re-suspended in 1mL HBSS (for a final lipid concentration of about 10 mg/mL) and vortexedfor 2-3 minutes. Subsequently, the lipid suspension was extruded usingan Avanti mini-extruder constructed with one 100-nm pore size membraneto form small unilamellar vesicles at 10 mg/mL.

1. Dosing of Antibodies in Cells

The day before assay, human macrophage cells or HEK293-H6 cells wereplated at 100,000 cells/well or 40,000 cells/well, respectively, on a96-well plate coated with poly-D-lysine. Antibodies were diluted intoPBS starting at 300 nM and proceeding in a 10-point serial dilutiontitration with 3-fold dilutions between points. For antagonistdose-response curves, lipid vesicles containing 70% DOPC and 30% POPS at1 mg/mL final concentration were also included in the antibody/PBSmixture. The cells were washed 3 times with HBSS using a Biotek 405/406plate washer, after which 50 μL per well of the antibody/PBS (with orwithout vesicles) solution was added using a Hamilton Nimbus liquidhandler. The cell plate was then transferred to a 37° C. incubator for 5minutes. The liposome/antibody solution was removed by flicking theplate, and 40 μL lysis buffer (Cell Signaling Technologies, CST)containing 1 μM PMSF was added using the liquid handler. The lysate wasthen either frozen at −80° C. or immediately assayed in the AlphaLisaassay.

Human macrophage cells were prepared for assay as follows. Humanmonocytes were isolated following the RosetteSep human monocyteenrichment cocktail protocol (Stemcell Technologies, REF #15068) fromfresh blood. Isolated monocytes were washed in wash buffer (PBS+2% FBS)and resuspended in 10 mL ACK lysis buffer (ThermoFisher Scientific,Catalog No. A10492) to lyse red blood cells. Twenty (20) mL of washbuffer was added to stop cell lysis, and the sample was centrifuged andwashed once more with culture media (RPMI, 10% Hyclone FBS, 1% SodiumPyruvate, 1% Glutamax, 1% non-essential amino acids, and 1%Penicillin-streptomycin). Human monocytes were then differentiated intomacrophage cells in culture media in the presence of 50 ng/mL humanrecombinant M-CSF (Gibco, Catalog No. PHC9501) at 250-mL flask. Freshhuman M-CSF was spiked on day 3 and human macrophages were subsequentlyharvested on day 5 and used for assay.

2. AlphaLisa Assay

Cell lysates were assayed for pSyk using the standard protocol for thePerkin Elmer pSyk AlphaLisa kit. In brief, 10 μL of lysate/well wastransferred to a white opaque 384 well Optiplate (Perkin Elmer). Next, 5μL of Acceptor Mix (containing the working solution of acceptor beads)was added per well, followed by sealing of plates with foil seals andincubation for 1 hour at room temperature. Subsequently, 5 μL of DonorMix (containing the working solution of donor beads) was added to eachwell under reduced light conditions. Plates were again sealed andincubated for 1 hour at room temperature. Finally, the plates were readusing AlphaLisa settings on a Perkin Elmer EnVision plate reader.

FIG. 2 illustrates representative anti-TREM2 antibody dose responsecurves for pSyk signal activation in primary human macrophage cells.Solid black circles (•) represent anti-TREM2 antibody, and open whitecircles (∘) represent isotype control. Each curve represents the mean ofthree independent experiments, and EC₅₀ values are provided in Table 1below. The results indicate that anti-TREM2 antibodies are able toactivate TREM2-DAP12 ITAM signaling in primary human macrophages.

Liposome Response Assay in iPSC Microglia

TREM2 agonist antibodies and phosphotidylserine-containing liposomesactivate pSyk via TREM2. In order to understand the effect of anti-TREM2antibodies on Syk signaling in the presence of liposomes, iPSC microgliawere pre-treated with anti-TREM2 antibody, followed by assessment of theliposome response in the cells.

Prior to assay, iPSCs were first differentiated into hematopoieticprogenitor cells (HPCs) using a commercially available kit (STEMdiffHematopoietic Kit from StemCell Technologies). HPCs were transferred toa plate containing primary human astrocytes and co-cultured for 14-21days. Once floating cells in co-culture were predominantly identified asmature microglia (>80%), the microglia were used for assay.

Two days prior to assay, human iPSC microglia were plated at 30,000cells/well on a 96-well plate coated with poly-D-lysine. Antibodies werediluted to 100 nM into media containing IMDM, 10% Hyclone FBS, and 1%Pen-strep, and the cells were dosed with the antibody solution for 24hours or 5 minutes at 37° C. Subsequently, the cells were washed oncewith HB SS and then dosed with lipid vesicles containing 70% DOPC and30% POPS at 1 mg/mL for 5 minutes at 37° C. The liposome solution wasremoved by flicking the plate, and 30 μL lysis buffer (Cell SignalingTechnologies, CST) containing 1 μM PMSF was added. The lysate was theneither frozen at −80° C. or immediately assayed in the AlphaLisa assay.The cell lysates were assayed for pSyk using the standard protocol forthe Perkin Elmer pSyk AlphaLisa kit as described above.

FIGS. 3A and 3B illustrate the activation of pSyk signal in human iPSCmicroglia incubated with anti-TREM2 antibodies, followed by dosing withlipid vesicles and assessment of the liposome response in the cells.White bars indicate incubation with PBS instead of lipid vesicles as acontrol. The data represent the mean and standard error of 2-7independent experiments. FIG. 3A illustrates data for iPSC microgliapre-treated with antibody for 5 minutes, and FIG. 3B illustrates datafor iPSC microglia pre-treated with antibody for 24 hours. The resultsshow that pre-treatment of human iPSC microglia with anti-TREM2antibodies produces an increase in the phospho-Syk signal elicited byliposomes compared to isotype control, indicating that the anti-TREM2antibodies do not interfere with, but instead enhance, lipid activationof pSyk signaling in cells.

Human TREM2 NFAT Reporter Assay

Human TREM2/DAP12-expressing Jurkat NFAT cell lines were generated asfollows. Jurkat NFAT reporter cells were infected with lentiviral vectorexpression of human TREM2 and DAP12 and cultured in RPMI containing 10%Hyclone FBS and 1% penicillin/streptomycin. Stable expressing cloneswere selected in the presence of puromycin and Zeocin. The cell surfaceTREM2 expression was evaluated by flow cytometer using a biotinylatedanti-TREM2 antibody (SEQ ID NOS:66 and 67). The clone that illustratedthe highest wild type TREM2 expression level was selected and named ashTrem2/NFAT Jurkat reporter cells for the assay described below.

The day prior to assay, 96-well plates were pre-coated with anti-TREM2antibody or isotype control at a 0-500 nM dose titration (45 μL/well,total 12 points) and incubated overnight at 4 C. After overnightincubation, the pre-coated plate was washed twice with PBS and thenloaded with hTrem2/NFAT Jurkat reporter cells (10⁶ cells/well) in 200 μLfresh culture media (RPMI with 10% Hyclone FBS and 1%penicillin/streptomycin). The plate was incubated at 37° C. for 24hours, after which 50 μL/well of quantlucia solution were added to eachwell and mixed well. For analysis, 20 μL of solution were removed fromeach well and transferred to a 384-well white plate for measurement ofsignal by luminometer (Perkin Elmer Envision).

FIG. 4 includes representative anti-TREM2 antibody dose-response curvesfor activation of NFAT as measured by detection of the reporter geneluciferase, and EC₅₀ values for activation are provided in Table 1below. The results in FIG. 4 illustrate that relative to isotypecontrol, candidate anti-TREM2 antibodies were capable of inducing NFATactivation and sufficient downstream signaling to activate atranscriptional response.

Survival Assay in Human Macrophage Cells

Human monocytes were isolated following the RosetteSep human monocyteenrichment cocktail protocol (Stemcell Technologies, Catalog No. 15068).Isolated monocytes were washed in wash buffer (PBS+2% FBS) andresuspended in 10 mL ACK lysis solution (ThermoFisher Scientific,Catalog No. A10492) to lyse red blood cells. Twenty (20) mL wash bufferwas added to stop lysis. The cell suspension was centrifuged and washedonce with culture media (RPMI 1640+10% FBS+penicillin/streptomycin).Cells were resuspended in culture media at a density of 10⁶ cells μL/mLand used in the survival assay described below.

The day prior to assay, 96-well plates were pre-coated with anti-TREM2antibody or isotype control at a 0-200 nM dose titration (45 μL/well,total 12 points) and incubated overnight at 4° C. After overnightincubation, the pre-coated plate was washed twice with PBS and thenloaded with human monocyte (10⁵ cells/well) in the presence of lowconcentration human M-CSF (5 ng/mL, Gibco, Catalog No. PHC9501). After 5days at 37° C., the media was aspirated, and 100 μL PBS+100 μLCelltiter-glo media (Promega, Catalog No. G7571) was added to each well.After 10 minutes of incubation, the cell media was transferred tomultiwell plates compatible for luminometer use, and luminescence forcell viability was recorded.

FIG. 5 illustrates representative anti-TREM2 antibody dose-responsecurves of cell survival in human macrophage cells under low M-CSFconditions, and EC₅₀ values for survival are provided in Table 1 below.The results indicate that TREM2 agonist antibodies have sufficientreceptor activating capacity to induce a transcriptional response formodulating cellular function and promoting survival of human macrophagecells under low M-CSF conditions.

Biacore Kinetic Measurement of Antibodies

Surface plasmon resonance (Biacore™ 8K instrument) was used to measureanti-TREM2 antibody affinities for human and cynomolgus TREM2 ECD.Anti-TREM2 antibodies were captured using Human Fab Capture Kit (GEHealthcare Life Sciences, Catalog No. 28958325) on a Biacore Series SCM5 sensor chip (GE Healthcare Life Sciences, Catalog No. 29149604).Serial 3-fold dilutions of recombinant human or cynomolgus TREM2 wereinjected at a flow rate of 30 μL/min. Antibody binding was monitored for300 seconds, followed by monitoring of antibody dissociation for 600+seconds in HBS-EP+ running buffer (GE Healthcare Life Sciences, CatalogNo. BR100669). The binding response was corrected by subtracting the RUvalue from a blank flow cell. A 1:1 Languir model of simultaneousfitting of k_(on), and k_(off) was used for kinetics analysis. K_(D)binding values are provided in Table 1 below.

TABLE 1 In Vitro Characteristics of Antibodies pSyk Biacore activationpSyk Cell binding NFAT Survival Biacore cyno (human activation (humanEC50 (nM)* EC50 (nM)* hTREM2 TREM2 macrophages) (HEK293-H6) macrophages)*Plate *Plate Antibody K_(D)(nM) K_(D)(nM) EC50 (nM) EC50 (nM) EC50 (nM)coated mAb coated mAb CL0020123 0.068 5.7  7 ± 2.7 0.3 Not 44.7 ± 30.3Calculable CL0020188 5.2 2.0 7.7 ± 1.5 0.5 91.2 ± 47.0 2.4 ± 1.3 NB: nobinding detected ND: not determined

Example 2. Modulation of Soluble TREM2 Levels and Phagocytosis Behaviorin Human Macrophage Cells Soluble TREM2 Dose Response Assay in HumanMacrophages

Human macrophage cells were generated as described above. One day priorto assay, human macrophage cells were plated at 100,000 cells/well on a96 well plate coated with poly-D-lysine. Antibodies were diluted inhuman macrophage media (RPMI, 10% Hyclone FBS, 1% Sodium Pyruvate, 1%Glutamax, 1% non-essential amino acids, and 1% Penicillin-streptomycin)starting from 300 nM and proceeding in a 10-point serial dilutiontitration with 3-fold dilutions between points. The cells were dosedwith the antibodies and incubated for 24 hours. After incubation withthe antibodies, the plate was spun down to remove debris, and thesupernatants collected for soluble TREM2 measurement.

Soluble TREM2 was measured as follows. Briefly, MSD small spotstreptavidin plates (Meso Scale Discovery) were coated with biotinylatedanti-hTREM2 polyclonal antibody (R&D Systems) overnight at 4° C. Theplates were then blocked with 3% BSA/TBST for 1 hour at roomtemperature. Samples and standards were prepared by heating to 95° C.for 5 minutes in SDS-containing buffer. The prepared samples andstandards were diluted 1:10 in 3% BSA/TBST in the assay plate afterblocking. A TREM2-His protein diluted in 3% BSA/TBST was used as astandard for absolute quantification. Following a two-hour incubation atroom temperature, the plates were washed with TBST. The primarydetection antibody, sulfo-tagged goat anti-human TREM2 (R&D Systems),was diluted in 3% BSA/TBST, added to the plates, and incubated for onehour at room temperature. After washing with TBST, the MSD plates weredeveloped using 2×MSD read buffer T, followed by detection using an MSDSector plate reader. MSD values were converted to absolute quantities ofsTREM2 by fitting a standard curve using Prism 7.0 software (Graphpad).Modulation of TREM2 shedding was represented as a ratio of soluble TREM2from cells incubated with test anti-TREM2 antibodies normalized tosoluble TREM2 from cells cultured with no specific anti-TREM2 antibodyin the media.

FIG. 6 illustrates representative soluble TREM2 levels (sTREM2) as afunction of the anti-TREM2 antibody concentration. The results indicatethe anti-TREM2 antibodies are capable of decreasing sTREM2 levels inhuman macrophage cells in a dose-dependent manner after overnighttreatment.

Phagocytosis Assay in Human Macrophages

Human macrophage cells were generated as described above. Two days priorto assay, human macrophage cells were plated at 80,000 cells/well on a96 well plate coated with poly-D-lysine. Antibodies were diluted at 100nM into media containing RPMI, 10% Hyclone FBS, 1% Sodium Pyruvate, 1%Glutamax, 1% non-essential amino acids, and 1% Penicillin-streptomycin.The cells were then dosed with antibody solution for 24 hours at 37° C.The cell nuclei and cell membrane were then stained for 10 minutes,after which pHrodo-myelin was added at 5 μg/mL. The cells were thenincubated for 4 hours at 37° C. The pHrodo fluorescence was measured percell on a high content confocal microscope (Opera Phoenix), and thefluorescence intensity quantified on the instrument software.

pHrodo-myelin was prepared by purifying myelin from wildtype C57Bl/6mouse brain (Jackson Laboratories) using methods described in Safaiyanet al. (2016, Nature Neuroscience 19(8):995-998). Followingpurification, myelin was resuspended in PBS and adjusted to 1 mg/mLprotein concentration using the DC Protein Assay Kit 2 (BioRad, CatalogNo. 5000112). Myelin was tagged with pHrodo-red using a microscalelabeling kit (ThermoFisher, Catalog No. P35363) according tomanufacturer instructions. Excess label was removed by pelleting themyelin at 10,000 g for 5 min, removing the supernatant, and repeatingthese steps 3-5 times.

FIG. 7 illustrates representative results of the phagocytosis assay inhuman macrophage cells. Myelin phagocytosis was measured by detectingand quantifying pHrodo fluorescence in microscopic images ofTREM2-treated macrophage cells and comparing the measured values tothose of an isotype control. The results show that human macrophagestreated with exemplary TREM2 agonist antibodies increase pHrodo-myelinphagocytosis relative to isotype control, indicating that the anti-TREM2antibodies can enable beneficial clearance of myelin debris in cells.

Example 3. Modulation of Lipid Accumulation in iPSC Microglia LipidStorage Assay

Prior to assay, iPSCs were first differentiated into hematopoieticprogenitor cells (HPCs) using a commercially available kit (STEMdiffHematopoietic Kit from StemCell Technologies). HPCs were transferred toa plate containing primary human astrocytes and co-cultured for 14-21days. Once floating cells in co-culture were predominantly identified asmature microglia (>80%), the microglia were used for assay.

Cells (iPSC microglia, 30,000 cells/well) were plated on PDL-coated96-well plates in full serum media. After 24 hours at 37° C., purifiedunlabeled myelin (50 μg/mL final concentration, purified from wildtypeC57Bl/6 mouse brain (Jackson Laboratories) using methods described inSafaiyan et al. (2016, Nature Neuroscience 19(8):995-998)) was spikedinto the wells. After 24 hours at 37° C. of lipid treatment, anti-TREM2antibody or RSV control was spiked into the wells to a finalconcentration of 100 nM. The cells were incubated for another 48-72hours at 37° C. before collecting or imaging cells. For myelin washoutexperiments, myelin was removed after the 24-hour incubation period andreplaced with antibody-containing media for a subsequent 24-48 hours ofincubation.

For Nile Red imaging, the supernatant was removed, and cells wereincubated at 37° C. for 30 minutes in live cell imaging buffer (LifeTechnologies, Catalog No. A14291DJ) containing 1 μM Nile Red(ThermoFisher, Catalog No. N1142) and 1 drop/mL of Nucblue(ThermoFisher, Catalog No. R37605). After the incubation period, thestaining solution was removed, and the cells were fixed in 4%paraformaldehyde. The cells were then imaged using Alexa 568 and DAPIillumination settings on an Opera Phoenix high content confocal imager.Lipid spots were analyzed using a spot-finding algorithm on the Harmonysoftware supplied with the instrument. FIG. 8A includes a representativemicroscopy image of iPSC microglia treated with either vehicle or myelin(50 μg/mL final concentration) for 24 hours, followed by incubation withan isotype control or an exemplary anti-TREM2 antibody (CL0020123) for72 hours. FIG. 8B is a representative bar chart for the same anti-TREM2antibody used in the microscopy image of FIG. 8A. Quantification of NileRed staining was performed by total spot intensity per cell, and data isshown as the mean and standard deviation of three technical replicatesin different fields of the same microscopy sample.

For lipidomic analysis, cells were washed once with PBS while kept onice. A volume of 70 μL of a 9:1 methanol:water solution containing 1:100internal standards was added to the cells in the 96-well plate. Theplate was agitated on a shaker at 4° C. and 1200 rpm for 20 minutes andthen centrifuged for 5 minutes at 300×g. A 50 μL sample of supernatantwas transferred to LCMS vials and kept at −80° C. until analyzed on theinstrument.

Lipid levels were analyzed by liquid chromatography (Shimadzu Nexera ×2system, Shimadzu Scientific Instrument, Columbia, Md., USA) coupled toelectrospray mass spectrometry (QTRAP 6500+, Sciex, Framingham, Mass.,USA). For each analysis, 5 μL of sample was injected on a BEH C18 1.7μm, 2.1×100 mm column (Waters Corporation, Milford, Mass., USA) using aflow rate of 0.25 mL/min at 55° C. For positive ionization mode, mobilephase A consisted of 60:40 acetonitrile/water (v/v) with 10 mM ammoniumformate+0.1% formic acid; mobile phase B consisted of 90:10 isopropylalcohol/acetonitrile (v/v) with 10 mM ammonium formate+0.1% formic acid.For negative ionization mode, mobile phase A consisted of 60:40acetonitrile/water (v/v) with 10 mM ammonium acetate; mobile phase Bconsisted of 90:10 isopropyl alcohol/acetonitrile (v/v) with 10 mMammonium acetate. The gradient was programmed as follows: 0.0-8.0 minfrom 45% B to 99% B, 8.0-9.0 min at 99% B, 9.0-9.1 min to 45% B, and9.1-10.0 min at 45% B. Electrospray ionization was performed in eitherpositive or negative ion mode applying the following settings: curtaingas at 30; collision gas set at medium; ion spray voltage at 5500(positive mode) or 4500 (negative mode); temperature at 250° C.(positive mode) or 600° C. (negative mode); ion source Gas 1 at 50; ionsource Gas 2 at 60. Data acquisition was performed using Analyst 1.6.3(Sciex) in multiple reaction monitoring mode (MRM), with the followingparameters: dwell time (msec) and collision energy (CE); declusteringpotential (DP) at 80; entrance potential (EP) at 10 (positive mode) or−10 (negative mode), and collision cell exit potential (CXP) at 12.5(positive mode) or −12.5 (negative mode). Lipids were quantified using amixture of non-endogenous internal standards. Lipids were identifiedbased on their retention times and MRM properties of commerciallyavailable reference standards (Avanti Polar Lipids, Birmingham, Ala.,USA).

FIGS. 8C and 8D illustrate levels of cholesterol ester (CE) (FIG. 8C)and triacylglyceride (TAG) lipid species (FIG. 8D) as detected by massspectrometry in cell lysates of iPSC microglia cells treated withexemplary anti-TREM2 antibodies for 72 hours after a 24-hour myelintreatment. FIGS. 8E and 8F illustrate levels of cholesterol ester (CE)(FIG. 8E) and triacylglyceride (TAG) lipid species (FIG. 8F) as detectedby mass spectrometry in cell lysates of iPSC microglia cells whichunderwent myelin washout experiments with exemplary anti-TREM2antibodies. LC/MS data generated in FIGS. 8C-8F were normalized to theinternal standards for CE data and normalized to myelin+ isotype controlfor each individual lipid species for TAG data.

Lipid accumulation in iPSC microglia is induced by myelin treatment,which is reflected by an increase in neutral lipid staining (Nile Red)and by LC/MS for detection of specific lipid species in cellularlysates. The data illustrated in FIGS. 8A-8F collectively indicate thattreatment of iPSC microglia cells post-myelin challenge with theexemplary anti-TREM2 antibodies reduced accumulation of lipid species,as indicated by the decrease in neutral lipid staining in cells and bythe decrease of CE and TAG lipid species levels measured by LC/MS. Thereduction of lipid levels as a result of antibody treatment was observedat different timepoints ranging from 24 hours to 72 hours. To eliminatethe possibility that the reduction in lipid levels is caused by blockingof lipid uptake, myelin washout experiments in which myelin was removedprior to anti-TREM2 antibody addition were carried out. FIG. 8Fillustrates that anti-TREM2 antibodies also reduced lipid levels in iPSCmicroglia with myelin washout prior to antibody treatment relative toisotype control.

Example 4. Functional Epitope Binning of Antibodies

TREM2 antibody epitope bins were determined by competition binding onTREM2 protein. Epitope binning experiment was performed on a CarterraLSA instrument using a classical sandwich epitope binning configurationmethod at 25° C. All test antibodies were immobilized onto a HC30M chipby amine coupling. Multiple cycles of sandwich competition binding werethen carried out for the test antibodies. Each cycle consisted ofantigen (His-tagged TREM2 ECD) injection followed by analyte antibodyinjection to the immobilized antibodies. At the end of each cycle, thesurface of immobilized antibodies were regenerated by injecting a low pHbuffer (pH=3) containing 1.25 M NaCl. Epitope binding data was evaluatedby Carterra software to create competition matrix and epitope bins.Results are provided in Table 2.

Two agonist bins were identified by epitope binning of the anti-TREM2antibodies: (1) stalk binding agonists, and (2) IgV domain bindingagonists. Antibodies within the same bins demonstrated the samefunction, e.g., inhibition of TREM2-DAP12 pSyk activation by lipidligand (antagonist antibodies), activation of pSyk with antibody alone(stalk binding agonists, IgV domain binding agonists).

TABLE 2 Anti-TREM2 Antibody Bins, Annotated by Functional Class Bin 1Bin 2 Stalk Agonist IgV Agonist CL0020188 CL0020123

Example 5. Antibody Binding to TREM2 Stalk Peptide

TREM2 antibodies were evaluated for binding to human and mouse TREM2stalk region peptides. The tested peptides included: (1) full lengthstalk region (amino acids 129-172 of human TREM2, UniProtKB Q9NZC2;amino acids 131-169 of mouse TREM2, UniProtKB Q99NH8) and (2) atruncated stalk peptide containing the ADAM10/17 cleavage site (aminoacids 149-163 of human/mouse TREM2). Antibody binding to TREM2 stalkpeptides was detected using standard sandwich ELISA. Briefly, a 96-wellhalf-area ELISA plate was coated with streptavidin overnight at 4° C.The following day, biotinylated TREM2 stalk peptides diluted in 1%BSA/PBS were added to the plate and incubated for 1 hour. Antibodiesdiluted in 1% BSA/PBS were then added and incubated for 1 hour.Antibodies bound to peptide were detected with anti-human kappa-HRPsecondary antibody (Bethyl Laboratories, Inc.) diluted in 1% BSA/PBS.Plates were assayed by reaction with a detection reagent (One-step TMBUltra, Thermo) and measurement of absorbance at 450 nm (A450) bystandard spectrophotometry instrumentation (BioTek®). The results areprovided in Table 3, below. Data are shown as normalized values (foldover background where background=isotype control).

TABLE 3 Anti-TREM2 Antibody Binding to TREM2 Stalk Peptides IsotypeCL0020188 CL0020123 Human TREM2 peptide binding 1.0 3.0 1.2 (aa 149-163)Human TREM2 peptide binding 1.0 51 1.2 (aa 129-172) Mouse TREM2 peptidebinding 1.0 0.9 0.9 (aa 149-163) Mouse TREM2 peptide binding 1.0 33 1.0(aa 131-169)

Table 3 depicts human and mouse stalk region peptide-antibody bindinginteractions which support the epitope binning data in Table 2. Based onthe data in Table 3, the site at which certain anti-TREM2 antibodiesappear to bind corresponds to amino acids 129-148 in the TREM2extracellular stalk region.

The ability of anti-TREM2 antibodies to inhibit TREM2 stalk peptidecleavage by ADAM17 was also analyzed using a fluorescence polarizationassay. TREM2 stalk peptides were first prepared in assay buffer (25 mMTris pH 7.5, 2.5 μM ZnCl₂, 0.005% Brij-35) with streptavidin. Anti-TREM2antibodies were then pre-incubated with TREM2 stalk peptides for 30minutes at room temperature. After the pre-incubation period, ADAM17(R&D systems, Catalog No. 930-ADB) was added and incubated with thepeptides for 20 hours at 37° C. The following day, samples were furtherdiluted in assay buffer and transferred to a black opaque 384-wellplate. Fluorescence polarization was subsequently measured on PerkinElmer EnVision plate reader. The fluorescence polarization of TREM2stalk peptides pre-incubated with anti-TREM2 antibody was compared tofluorescence polarization of full-length TREM2 stalk peptide and enzymecontrol (full-length TREM2 stalk peptide with ADAM17).

TREM2 stalk-binding antibodies significantly increased fluorescencepolarization, demonstrating partial inhibition of stalk peptide cleavageby ADAM17 (clones CL0020141, CL0020188, CL0020313, CL0020308). IgVbinding antibody CL0020107 did not bind TREM2 stalk region peptide andthus did not show an effect on peptide cleavage in the fluorescencepolarization assay.

Example 6. Pharmacokinetic Analysis of Anti-TREM2 Antibodies

The pharmacokinetic profiles of the anti-TREM2 antibodies were evaluatedin mice. C57BL/6J mice were purchased from Jackson Laboratory (Stock No.000664) at 2 months of age used for a 7-day pharmacokinetic (PK) studyand target engagement study. Human Trem2 cDNA KI homozygous mice(huTrem2^(KI/KI)) were used for 24-hour target engagement study at 3months of age. Generation and breeding of the human Trem2 cDNA KI miceare described below.

Generation of Human Trem2 cDNA KI Mouse Model

A human TREM2 cDNA KI mouse (huTrem2^(KI/KI)) was generated as follows.Human Trem2 cDNA-pA sequence was inserted at the mouse Trem2 endogenousATG start site. The insertion of human Trem2 cDNA-pA resulted inreplacement of the exonl sequence of mouse Trem2, which allowsexpression of human Trem2 cDNA driven by the endogenous mouse promoterand disruption of the expression of endogenous mouse Trem2. ThehuTrem2^(KI/KI) mouse was generated in C57BL/6 genetic background usinghomologous recombination.

For the huTrem2^(KI/KI) targeting vector, the long homology arm (LA)extends about 3.6 kb upstream of the 5′ end of the human Trem2 cDNA-pAsequence, and the short homology arm (SA) extends about 2.3 kbdownstream of the 3′ to the FRT-flanked Neo cassette. Both the long andshort homology arms were amplified from a C57BL/6 BAC clone (RP23:358G22) and then sub-cloned into a ˜2.4 kb pSP72 (Promega) backbonevector containing an ampicillin selection cassette. A hUBS-gb2FRT-flanked Neomycin cassette was inserted immediately downstream of thehTrem2-pA cassette, resulting in a targeting vector of about 13.6 kb insize. Ten (10) μg of the targeting vector was linearized withrestriction enzyme Not I (New England Biolabs) and then transfected intoFLP C57Bl/6 (B6) embryonic stem (ES) cells by electroporation. Afterselection with G418 antibiotic, surviving clones were expanded for PCRanalysis to identify positive recombinant ES clones. The primersequences for PCR screening (SEQ ID NO:53=5′-AGG AAT GTG GGG AGC ACGGAG-3′ and SEQ ID NO:54=5′-TGC ATC GCA TTG TCT GAG TAG GTG-3′) amplifieda 2.81-kb fragment containing the region from the bghpA element to thedownstream of the short homology arm (SA) outside the 3′ region. Fiveclones were identified as positive and selected for further expansion.The Neo cassette was removed by Flp transgene during ES clone expansion.

Genomic DNA extracted from the five positive clones were firstcharacterized by sequencing analysis. A 1.19-kb product was amplifiedand sequenced by primers (SEQ ID NO:55=5′-ACC CTA GTC CTG ACT GTT GCTC-3′; SEQ ID NO:56=5′-TAT AGG AAC TTC GCG ACA CGG ACA C-3′) to confirmthe 5′ genome/neo cassette junction and 3′ KI cassette junctions. Thesequencing results confirmed the introduction of human Trem2 cDNA-pAsequence in all of the five clones.

The five positive clones were further characterized by Southern Blottinganalysis using probes targeted against short arm and long arm. ES cellgenomic DNA digested with Ssp I and Bam HI were hybridized with shortarm and long arm probe respectively. All five ES clones were confirmedto carry the correct homologous recombination events in both long andshort arms. The primer sequences for amplifying the short arm probe (658bp) are (SEQ ID NO:57=5′-ACA GGA GGG ACC TAC CTT CAG 3′; SEQ IDNO:58=5′-GCC TGC CTT TCA GAG ACC TCA GTC-3). The primer sequences foramplifying the long arm probe (681 bp) are (SEQ ID NO:59=5′-CCT CTC CGGCTG CTC ATC TTA CTC-3′; SEQ ID NO:60=5′-GTC TCT CAG CCC TGG CAG AGTTTG-3′).

All five ES cell clones were then injected into C57BL/6 blastocysts. Thepups from one clone were confirmed with germline transmission by PCRgenotyping. The primers for genotyping are (SEQ ID NO:61=pr1: 5′-CGC CTACCC TAG TCC TGA CTG TTG-3′, SEQ ID NO:62=pr2: 5′-AAA GCC TAC AGC ATC CTCACC TC-3′; and SEQ ID NO:63=pr3: 5′-GCA TCA TGG GGT TGT AGA TTC CG-3′).The PCR product for the pr1/pr2 on the wild-type is 658 bp. The PCRproduct for pr1/pr3 on the KI allele is 469 bp.

Antibody Dosing and Plasma/CSF Collection

For PK analysis, the C57BL/6J mice were dosed with anti-TREM2 antibodiesor control IgG at 10 mg/kg through intravenous (IV) tail vein injection(dosing volume of about 200 μL/mouse, n=3 for each group). Blood sampleswere collected at 1 hour, 24 hours, 4 days, and 7 days after dosing. Theblood samples at the first three time points were collected throughsubmandibular bleeding by using 3-mm lancets (GoldenRod animal lancets).The last and terminal blood sample at day 7 was collected throughcardiac puncture. Blood was collected in EDTA tubes (Sarstedt Microvette500 K3E, Catalog No. 201341102), inverted slowly to mix, and centrifugedat 4° C. The plasma (top) layer was transferred to 1.5-mL Eppendorftubes and stored at −80° C. until analysis.

For the 24-hour target engagement study, C57BL/6J mice were used to testmouse surrogate anti-TREM2 antibodies, and huTrem2^(KI/KI) mice wereused to test human anti-TREM2 antibodies. The mice were dosed withanti-TREM2 antibodies or control IgG at 100 mg/kg through intravenous(IV) tail vein injection (dosing volume of about 200 μL/mouse, n=5 foreach group). Blood samples were collected 24 hours before dosing todetermine baseline levels of TREM2. Terminal blood and CSF samples werecollected 24 hours after dosing. Plasma preparation was prepared asdescribed above. For collection of the CSF sample, a sagittal incisionwas made at the back of the skull, and subcutaneous tissue and muscleswere separated to expose the cisterna magna. A pre-pulled glasscapillary tube was used to puncture the cisterna magna and collect theCSF sample. The CSF was then centrifuged at 4° C. to remove bloodresidue, and the CSF supernatant was transferred to a 0.5-mL Low ProteinLoBind Eppendorf tube (Eppendorf, Catalog No. 022431064) for storage at−80° C. until analysis.

Analysis of In Vivo Anti-TREM2 Antibody Plasma Levels

For anti-TREM2 antibody PK analysis, total antibody concentrations inmouse plasma were quantified using a generic human anti-Fc sandwichELISA. A 384-well MaxiSorp plate was coated overnight with 1 μs/mLanti-huFc donkey polyclonal (Jackson Immunoresearch). Followingincubation with plasma diluted 1:2,000 or 1:20,000 in assay buffer(PBST, 1% BSA), an anti-huFc donkey antibody conjugated to HRP (JacksonImmunoresearch) was added as the detection reagent. Standard curves weregenerated for each individual antibody, from 2 nM to 2.7 pM using 3-folddilutions, using a five-parameter logistic regression.

FIG. 9 illustrates representative mouse PK profiles for certainanti-TREM2 antibodies. Antibody clearance rates (CL [mL/day/kg]) areprovided for each represented antibody over a 7-day period and arecompared to normal effectorless isotype control. Each antibodyillustrated in FIG. 9 exhibited comparable clearance rates relative toisotype control.

In Vivo Target Engagement: sTREM2 Plasma Levels

For measurement of soluble TREM2 (sTREM2) plasma levels, human TREM2cDNA KI mice (huTrem2^(KI/KI)) were bled and then intravenously treatedwith 100 mg/kg of test anti-TREM2 antibody or isotype control. Mice werebled 24 hours post-dosing. Plasma were obtained from blood samples andevaluated in an MSD assay conducted as follows. MSD SECTOR Plates werecoated with 1 μg/mL capture antibody (R+D anti-TREM2 antibody, CatalogNo. MAB17291-100) diluted in PBS and incubated overnight at 4° C. Thesample wells were blocked for 1 hour with undiluted MSD Blocker A.Plasma samples were diluted 1:20 in 25% MSD Blocker A in Tris-bufferedsaline containing 0.05% Tween-20 (TBST) and added to each sample well onthe plate, which was then incubated for 2 hours at room temperature.Detection antibody (MSD sulfo-tagged goat anti-human, Catalog No.R32AJ-1, 1:1000) was subsequently added to each sample well, and theplate was incubated for 1 hour at room temperature. TBST washes wereperformed for each sample well with a Biotek plate washer. Detectionreagent (MSD Read buffer) was added and measured using a MSD Meso SectorS600 reader to obtain the results of sTREM2 binding to the antibodies.

FIGS. 10A and 10B illustrate total sTREM2 levels (FIG. 10A) andantibody-bound sTREM2 levels (FIG. 10B) in huTrem2^(KI/KI) plasma for anexemplary anti-TREM2 antibody. Data were normalized to pre-dose baselinelevels of sTREM2 for total and bound sTREM2 assays. The results indicatethat total circulating sTREM2 levels did not significantly changebetween mice treated with anti-TREM2 antibodies compared to isotypecontrol after 24-hour treatment with the antibodies, which suggests thattotal circulating sTREM2 levels are unaffected at early timepointspost-antibody dose. In contrast, antibody-bound sTREM2 levels werehigher in mice injected with anti-TREM2 antibodies relative to isotypecontrol.

Example 7. Sequence Optimization and Humanization of Anti-TREM2Antibodies

Exemplary anti-TREM2 antibodies were sequence optimized and humanized,followed by characterization for binding kinetics and bindingspecificity.

Sequence optimization was conducted by searching within CDR sequencesfor residues that are susceptible to chemical modification (e.g.,asparagine deamidation motifs (NG), aspartic acid isomerization motifs(DS), and potential oxidation residues (tryptophan (W) and methionine(M)) and making amino acid substitutions with conservative and germlineresidues to remove such sequence liabilities. Humanized andsequence-optimized variants of anti-TREM2 antibodies were then analyzedfor binding kinetics using Biacore and dose-titrated cell binding toHEK293-H6 cells (see, Example 1 for representative protocols).

Results for an analysis of the binding characteristics of humanized andsequence-optimized variants of antibody CL0020188 are provided in Table4. NG motifs in the CL0020188 CDR-H2 sequence (SEQ ID NO:5) and CDR-L1sequence (SEQ ID NO:7) were modified, grafted onto human frameworkregions, and analyzed. Table 4 provides K_(D) values as measured byBiacore and EC₅₀ values as measured by dose-titrated binding assay inHEK293-H6 cells.

TABLE 4 Binding Characteristics of Sequence-Optimized and HumanizedVariants of CL0020188 Clone hV_(H) hVL K_(D) EC₅₀ CL0020188-1 NG/graftNG/graft 2.3 nM 0.42 nM CL0020188-2 NG/3m NG/graft 3.4 nM 0.26 nMCL0020188-3 NG/graft TG/graft 6.8 nM 0.64 nM CL0020188-4 NG/3m TG/graft4.8 nM 0.44 nM CL0020188-5 NA/graft NG/graft 5.1 nM 0.45 nM CL0020188-6NA/3m NG/graft 4.0 nM 0.31 nM CL0020188-7 NA/graft TG/graft  10 nM 0.68nM CL0020188-8 NA/3m TG/graft 7.3 nM 0.51 nM Parent 9.5 nM 0.44 nM 3m =A24G/L45P/V48L in V_(H)

As illustrated in Table 4, humanized and sequence-optimized clones ofCL00201088 exhibited similar affinity values for hTREM2 compared to theparent antibody (K_(D)=9.5 nM), as measured by Biacore. This wasconsistent with cell-binding results in HEK293-H6 cells, which areillustrated in Table 4. Compared to the parent antibody (EC₅₀=0.44 nM),humanized and sequence-optimized clones exhibited comparable andsub-nanomolar affinity for TREM2 expressed in HEK293-H6 cells. Takentogether, the results indicate comparable binding kinetics between theparent antibody and the humanized and sequence-optimized variants.

Results for an analysis of the binding characteristics of humanized andsequence-optimized variants of antibody CL0020123 are provided in Table5. NG and DS motifs in the CL0020123 CDR-H2 sequence (SEQ ID NO:30) weremodified, grafted onto human framework regions, and analyzed. Table 5provides provides K_(D) values as measured by Biacore and EC₅₀ values asmeasured by dose-titrated binding assay in HEK293-H6 cells.

TABLE 5 Binding Characteristics of Sequence-Optimized and HumanizedVariants of CL0020123 Clone hV_(H) hVL K_(D) EC₅₀ CL0020123-1 NGDS (SEQID Vκ graft 0.14 nM 0.25 nM NO: 80)/2m CL0020123-2 NGDS (SEQ ID Vκ graft0.18 nM 0.23 nM NO: 80)/1m CL0020123-3 QGDS (SEQ ID Vκ graft 0.40 nM0.24 nM NO: 81)/2m CL0020123-4 NGES (SEQ ID Vκ graft 0.17 nM 0.17 nM NO:82)/2m CL0020123-5 QGES (SEQ ID Vκ graft 0.44 nM 0.37 nM NO: 83)/2mCL0020123-6 QGDS (SEQ ID Vκ graft 0.32 nM 0.29 nM NO: 81)/1m CL0020123-7NGES (SEQ ID Vκ graft 0.15 nM 0.19 nM NO: 82)/1m CL0020123-8 QGES (SEQID Vκ graft 0.39 nM 0.38 nM NO: 83)/1m Parent 0.10 nM 0.26 nM 1m = R71Ain V_(H) 2m = V67A/R71A in V_(H)

Compared to the parent antibody (K_(D)=0.10 nM), the humanized andsequence-optimized clones exhibited about 4-fold higher K_(D) values forbinding to hTREM2 as measured by Biacore. On the other hand, indose-titrated cell-binding assays in HEK293-H6 cells, the humanized andsequence-optimized clones exhibited comparable and sub-nanomolaraffinity for TREM2.

Example 8. In Vitro Characterization of Sequence-Optimized, HumanizedAnti-TREM2 Antibodies

Exemplary sequence-optimized and humanized anti-TREM2 antibodies(Example 7) were evaluated by in vitro methods as described in Examples1 and 3. The antibodies were assessed for TREM2 binding inTREM2-expressing HEK cells, TREM2-dependent pSyk signaling in HEK-H6cells, capability for promoting survival of human macrophage cells, andability to modulate lipid accumulation in iPSC microglia. FIGS. 11 to 14illustrate the results for representative anti-TREM2 antibodies. Theanti-TREM2 antibodies exhibited good cell binding in assays withTREM2-expressing HEK293-H6 cells (EC50 values of 0.34 nM (FIG. 11A) and0.08 nM (FIG. 11B). The anti-TREM2 antibodies also activated pSyksignaling in TREM2-expressing HEK293-H6 cells (FIGS. 12A and 12B). Inaddition, the anti-TREM2 antibodies induced macrophage survival, withbetter survival activity observed for a CL0020188 variant antibody (FIG.13). Finally, the anti-TREM2 antibodies demonstrated capability inreducing lipid accumulation in myelin-treated iPSC microglia (FIGS. 14Aand 14B).

Example 9. Mouse PK of Sequence-Optimized, Humanized Anti-TREM2Antibodies

The pharmacokinetic profiles of certain sequence-optimized, humanizedanti-TREM2 antibodies (Example 7) were evaluated in wild-type mice for a7-day pharmacokinetic (PK) study similar to that described in Example 6.Each dose group contained n=3 mice. Table 6 provides the PK propertiesof exemplary sequence-optimized and humanized anti-TREM2 antibodies(Example 7).

TABLE 6 Mouse PK Properties of Anti-TREM2 Antibodies Dose C₀ AUC_(0-inf)CL Clone (mg/kg) (μM) (μM*hr) (mL/day/kg) CL0020188 variant 10 1.14 ±0.05 207.78 ± 43.30 8.14 ± 1.80 CL0020188 variant 50 6.48 ± 0.61 236.49± 2.67  34.58 ± 0.39  CL0020123 variant 10 1.4999 223.1 13.2 Anti-BACEreference 10 1.017 60.19 33.3

Example 10. Cyno PK of Sequence-Optimized, Humanized Anti-TREM2Antibodies

The pharmacokinetic profiles of the anti-TREM2 antibodies were evaluatedin naïve cynomolgus monkeys. Naïve cynomolgus monkeys of 2-4 years ofage (approximately 2-3 kg in weight) were injected with anti-TREM2antibody via intravenous bolus injection. Administered doses included 3mg/kg and 25 mg/kg, with n=3 monkeys per dose group. Blood samples(about 1 mL) were collected pre-dose and at 10 minutes, 30 minutes, at1, 6, 12, and 24 hours, and at 3, 7, 10, 14, 17, 21, 24, and 28 dayspost-dose. The samples were chilled at about 5° C. prior tocentrifugation to obtain plasma, and the obtained plasma was maintainedon dry ice prior to storage at −70° C. until analysis. The plasmasamples were analyzed for anti-TREM2 antibody levels as follows.

For anti-TREM2 antibody PK analysis, the total antibody concentrationsin monkey plasma were quantified using a generic anti-human IgG sandwichelectrochemiluminescence immunoassay (ECLIA) on a Meso Scale Discovery(MSD) platform. Briefly, 1% casein-based PBS blocking buffer (ThermoScientific, MA) was added to an MSD GOLD 96-well small-spotstreptavidin-coated microtiter plate (Meso Scale Discovery, MD) andincubated for approximately 1 hr. Following the plate blocking and washsteps, a biotinylated anti-human_IgG goat antibody (SouthernBiotech, AL)at a working solution of 0.5 μg/mL was added to the assay plate andallowed to incubate for 1-2 hrs. Following the incubation and washsteps, plasma test samples (i.e., samples with anti-TREM2 humanizedantibodies) were added to the assay plate and incubated for 1-2 hrs.Note that the test samples must be diluted at the assayminimum-required-dilution (MRD) of 1:100 in 0.5% casein-based PBS assaybuffer (Thermo Scientific, MA), resulting in the final 1% plasma matrix,prior to adding to the assay plate. Following the capture of anti-TREM2antibody analyte and wash steps, a secondary ruthenylated (SULFO-TAG)anti-human IgG goat antibody (Meso Scale Discovery, MD) at a workingsolution of 0.4 μg/mL was added to the assay plate and incubated forapproximately 1 hr. Lastly, following the incubation and wash steps, anassay read buffer (1×MSD Read Buffer T) was added to the assay plate togenerate assay sample signals. The sample signal readouts from an MSDplate reader was in a form of electrochemiluminescence (ECL) signals andexpressed in ECL units (ECLU). All of the assay reaction stepspreviously described were done at the ambient temperature and withshaking on a plate shaker (where appropriate). The assay had a dynamiccalibration standard range of 19.5-2500 ng/mL (or 0.195-25 ng/mL inpost-MRD of 1:100) with 8 standard points serially-diluted at 1:2 plus ablank plasma sample. Plasma sample concentration was back-calculated offthe assay calibration standard curve which was fitted with a weighedfour-parameter non-linear logistic regression. Table 7 provides thepharmacokinetic (PK) properties of an exemplary sequence-optimized andhumanized anti-TREM2 antibody (Example 7).

TABLE 7 Cynomolgus PK Properties of Anti-TREM2 Antibodies DoseAUC_(0-inf) CL Clone (mg/kg) (μM*hr) (mL/day/kg) CL0020188 variant 25899.0 ± 53 4.46 ± 0.26 CL0020188 variant 3  61.2 ± 6.3 7.90 ± 0.77

The CL0020188 variant exhibited similar low clearance levels betweendifferent dose levels and a linear pharmacokinetic profile in cynomolgusmonkeys. In addition, there were no clinical pathology findings relatedto administration of the variant in cynomolgus monkeys (data not shown).

Example 11. Comparison of Anti-TREM2 Antibodies

The affinity of anti-TREM2 antibodies to human TREM2 and cynomolgusTREM2 were measured by Biacore (described in Example 1). The potency ofanti-TREM2 antibodies were measured in healthy human volunteer CSFsamples (Innovative Research) and healthy cynomolgus monkey CSF samples(Worldwide Primates), by MSD assay. The potency of each antibody wasdetermined by its EC50. Briefly, MSD GOLD 96-well small spotstreptavidin plates (MSD, Cat. No. L45SA) coated with capture antibody(biotinylated goat anti-human TREM2, R&D Systems, Cat. No. BAF1828) wasincubated with biofluid samples diluted 1:3 in Assay Buffer (25% (v/v)MSD Blocker A (MSD, Cat. No. R93BA-A), 75% (v/v) TBST) for one hour atroom temperature. After rinsing the wells with TBST, sulfo-taggedanti-TREM2 antibody was added in a serial dilution (4× dilution over 11points) to the wells of the plate and incubated for one hour at roomtemperature. The wells were washed with TBST, and MSD Read Buffer (MSD,Cat. No. R92TC-3) was added to the wells. Signal from the samples weremeasured using an MSD Meso Sector 5600 device. EC50 values for eachantibody was determined by four-parameter variable slope non-linearregression. Reference antibodies #1 and #2 correspond to 4C5 and 6E7described in WO 2018/195506. Reference antibody #3 corresponds toAL2p-58 described in WO 2019/028292. The variable regions of referenceantibody #1 are represented by SEQ ID NOS:73 and 74. The variableregions of reference antibody #2 are represented by SEQ ID NOS:75 and76. The variable regions of reference antibody #3 are represented by SEQID NOS:77 and 78. Results are provided in Table 8.

As illustrated in Table 8, the CL0020188 and CL0020123 variants asdisclosed herein, relative to reference antibodies, have strongeraffinity for human TREM2 and are more potent in binding sTREM2 in CSFsamples isolated from healthy human volunteer subjects. In addition, theCL0020188 variants, relative to reference antibodies, have strongeraffinity for cynomolgus TREM2 and are more potent in binding sTREM2 inCSF samples isolated from healthy cynomolgus monkey subjects. This isdemonstrated by the relative amount of antibody needed to reach the halfmaximal effective concentration for binding a given amount of sTREM2under the same conditions (EC50). As illustrated in Table 8, higherrelative amounts of reference antibodies #1, #2, and #3 are needed toachieve EC50 compared to the CL0020188 and CL0020123 antibodies.

TABLE 8 Comparison of Properties of Anti-TREM2 Antibodies K_(D) K_(D)EC50, bound EC50, bound (nM) (nM) sTREM2 in sTREM2 in Human Cyno HumanCSF Cyno CSF Antibody TREM2 TREM2 [nM] [nM] CL0020188-4 1.4 1.5 0.290.28 CL0020188-7 3.3 2.8 0.75 0.72 CL0020123-5 0.63 9.3 1.00 49.41CL0020123-7 0.08 9.4 0.54 16.88 Reference antibody 5.9 14 32.6 81.6 #1Reference antibody 4.3 9.2 7.4 6.5 #2 Reference antibody 8.7 9.2 7.2533.72 #3

Example 12. Epitope Mapping of Anti-TREM2 Antibodies

To identify the epitopes of the anti-TREM2 antibodies at the peptidelevel, hydrogen deuterium exchange (HDX) mass spectrometry was used.Recombinant human TREM2 alone or mixed with anti-TREM2 antibody wasincubated with deuterium oxide labeling buffer (50 mM sodium phosphate,100 mM sodium chloride, pH 7.0) for 0, 60, 600, and 3600 seconds at 20°C. Hydrogen/deuterium exchange was quenched by adding an equal volume of4 M guanidine hydrochloride, 0.85 M TCEP buffer (final pH 2.5). Thequenched samples were then subjected to pepsin/protease XIII digestionand LC-MS analysis. Briefly, the quenched samples were injected onto apacked pepsin/protease XIII column (2.1×30 mm) held at 20° C., and theresultant peptides were analyzed using an UPLC-MS system comprised of aWaters Acquity UPLC (Waters Corporation) coupled to a Q Exactive™HF-Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher). Thepeptides were separated on a 50 mm×1 mm C8 column held at −6° C. using a16.5-minute gradient from 2% to 31% solvent B (solvent B: 0.2% formicacid in acetonitrile; solvent A: 0.2% formic acid in water). The massspectra were recorded in MS only mode. Raw MS data was processed usingHDX Workbench (Pascal et al. 2012. Journal of The American Society forMass Spectrometry 23:1512-1521). The deuterium levels were calculatedusing the average mass difference between the deuterated peptide and itsnative form at tO. Peptide identification was carried out throughsearching MS/MS data against the human TREM2 sequence with Mascotsoftward (Matrix Science). The mass tolerance for precursor and productions were 10 ppm and 0.02 Da, respectively.

Based on the HDX mass spectrometry results, CL0020188 and variants ofCL0020188 bind to human TREM2 (SEQ ID NO:1) at amino acid residues143-149 (FPGESES (SEQ ID NO:69)), while CL0020123 and variants ofCL0020123 bind to human TREM2 at (i) amino acid residues 55-63(GEKGPCQRV (SEQ ID NO:70)), (ii) amino acids 96-107 (TLRNLQPHDAGL (SEQID NO:71)), and (iii) amino acid residues 126-129 (VEVL (SEQ ID NO:72)).

IX. Informal Sequence Listing

SEQ ID NO Sequence Description  1MEPLRLLILLFVTELSGAHNTTVFQGVAGQSLQVSCPYDSMKH Human TREM2 proteinWGRRKAWCRQLGEKGPCQRVVSTHNLWLLSFLRRWNGSTAITDDTLGGTLTITLRNLQPHDAGLYQCQSLHGSEADTLRKVLVEVLADPLDHRDAGDLWFPGESESFEDAHVEHSISRSLLEGEIPFPPTSILLLLACIFLIKILAASALWAAAWHGQKPGTHPPSELDCGHDPGY QLQTLPGLRDT  2EVKLLDSGGGLVQAGGSLRLSCAGSGFTFTDFYMSWIRQPPGKA CL0020306 V_(H)PEWLGVIRNKANGYTAGYNPSVKGRFTISRDNTQNILYLQMNTLRAEDTAIYYCARLSYGFDYWGQGVMVTVSS  3DIVMTQGALPNPVPSGESASITCQSSKSLLHSNGKTYLNWYLQR CL0020306 VLPGQSPQLLIYWMSTRASGVSDRFSGSGSGTDFTLKISSVEAEDVG VYYCQQFLEFPFTFGSGTKLEIK  4GFTFTDFYMS CL0020306 CDR-H1; CDR- H1 for CL0020188 andvariants CL0020188-1, CL0020188-2, CL0020188-3,CL0020188-4, CL0020188-5, CL0020188-6, CL0020188-7, and CL0020188-8  5VIRNKANGYTAGYNPSVKG CL0020306 CDR-H2; CDR- H2 for CL0020188 andvariants CL0020188-1, CL0020188-2, CL0020188-3, and CL0020188-4  6ARLSYGFDY CL0020306 CDR-H3  7 QSSKSLLHSNGKTYLN CL0020306 CDR-L1;CL0020307 CDR-L1; CL0020307-1 CDR-L1; CDR-L1 for CL0020188 andvariants CL0020188-1, CL0020188-2, CL0020188-5, and CL0020188-6  8WMSTRAS CL0020306 CDR-L2; CL0020307 CDR-L2; CL0020307-1 CDR-L2;CDR-L2 for CL0020188 and variants CL0020188-1, CL0020188-2, CL0020188-3,CL0020188-4, CL0020188-5, CL0020188-6, CL0020188-7, and CL0020188-8  9QQFLEFPFT CL0020306 CDR-L3; CL0020307 CDR-L3; CL0020307-1 CDR-L3 10EVKLLESGGGLVQPGGSLRLSCAASGFTFTNFYMSWIRQPPGRA CL0020307 V_(H)PEWLGVIRNRPNGYTTDYNPSVKGRFTISRDNTQNILYLQMSTLRADDTAFYYCTRLTYGFDYWGQGVMVTVSS 11DIVMTQGALPNPVPSGESASITCQSSKSLLHSNGKTYLNWYLQR CL0020307 VLPGQSPQLLIYWMSTRASGVSDRFSGSGSGTDFTLKISSVEAEVVG VYYCQQFLEFPFTFGSGTKLEIK 12GFTFTNFYMS CL0020307 CDR-H1 13 VIRNRPNGYTTDYNPSVKG CL0020307 CDR-H2 14TRLTYGFDY CL0020307 CDR-H3 15EVKLLDSGGGLVQAGGSLRLSCAGSGFTFTDFYMSWIRQPPGKA CL0020188 V_(H)PEWLGVIRNKANGYTAGYNPSVKGRFTISRDNTQNILYLQMNTLRAEDTAIYYCARLTYGFDYWGQGVMVTVSS 16DIVMTQGALPNPVPSGESASITCQSSKSLLHSNGKTYLNWYLQR CL0020188 VLPGQSPQLLIYWMSTRASGVSDRFSGSGSGTDFTLKISSVEAEDVG VYYCQQFLEYPFTFGSGTKLEIK 17ARLTYGFDY CDR-H3 for CL0020188 and variants CL0020188-1,CL0020188-2, CL0020188-3, CL0020188-4, CL0020188-5,CL0020188-6, CL0020188-7, and CL0020188-8 18 QQFLEYPFTCDR-L3 for CL0020188 and variants CL0020188-1, CL0020188-2, CL0020188-3,CL0020188-4, CL0020188-5, CL0020188-6, CL0020188-7, and CL0020188-8 19EVQLVESGGGLVQPGGSLRLSCAASGFTFTDFYMSWVRQAPGK CL0020188-1 V_(H);GLEWVSVIRNKANGYTAGYNPSVKGRFTISRDNSKNTLYLQMN CL0020188-3 V_(H)SLRAEDTAVYYCARLTYGFDYWGQGTLVTVSS 20DIVMTQTPLSLPVTPGEPASISCQSSKSLLHSNGKTYLNWYLQKP CL0020188-1 VL;GQSPQLLIYWMSTRASGVPDRFSGSGSGTDFTLKISRVEAEDVG CL0020188-2 VL;VYYCQQFLEYPFTFGQGTKVEIK CL0020188-5 VL; CL0020188-6 VL 21EVQLVESGGGLVQPGGSLRLSCAGSGFTFTDFYMSWVRQAPGK CL0020188-2 V_(H)GLEWVSVIRNKANGYTAGYNPSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLTYGFDYWGQGTLVTVSS 22DIVMTQTPLSLPVTPGEPASISCQSSKSLLHSTGKTYLNWYLQKP CL0020188-3 VL;GQSPQLLIYWMSTRASGVPDRFSGSGSGTDFTLKISRVEAEDVG CL0020188-4 VL;VYYCQQFLEYPFTFGQGTKVEIK CL0020188-7 VL; CL0020188-8 VL 23QSSKSLLHSTGKTYLN CDR-L1 for variants CL0020188-3, CL0020188-4,CL0020188-7, and CL0020188-8 24EVQLVESGGGLVQPGGSLRLSCAASGFTFTDFYMSWVRQAPGK CL0020188-5 V_(H);GLEWVSVIRNKANAYTAGYNPSVKGRFTISRDNSKNTLYLQMN CL0020188-7 V_(H)SLRAEDTAVYYCARLTYGFDYWGQGTLVTVSS 25 VIRNKANAYTAGYNPSVKGCDR-H2 for variants CL0020188-5, CL0020188-6, CL0020188-7, andCL0020188-8 26 EVQLVESGGGLVQPGGSLRLSCAGSGFTFTDFYMSWVRQAPGKCL0020188-6 V_(H); GPEWLSVIRNKANAYTAGYNPSVKGRFTISRDNSKNTLYLQMNCL0020188-8 V_(H) SLRAEDTAVYYCARLTYGFDYWGQGTLVTVSS 27EVQLQQSGAELVRSGASVKLSCTASGFSIEDFYIHWVKQRPEQG CL0020123 V_(H)LEWIGWIDPENGDSKYAPKFQGKATMTADTSSNTAYLHLSSLTSEDTAVYYCHADHGNYGSTMDYWGQGTSVTVSS 28DIQMNQSPSSLSASLGDTVTITCHASQHINVWLSWYQQKPGDHP CL0020123 VLKLLIYKASNLHTGVPSRFSGSGSGTGFTLTISSLQPEDIATYYCQQ GQTYPRTFGGGTKLEIK 29GFSIEDFYIH CDR-H1 for CL0020123 and variants CL0020123-1,CL0020123-2, CL0020123-3, CL0020123-4, CL0020123-5,CL0020123-6, CL0020123-7, and CL0020123-8 30 WIDPENGDSKYAPKFQGCDR-H2 for CL0020123 and variants CL0020123-1 and CL0020123-2 31HADHGNYGSTMDY CDR-H3 for CL0020123 and variants CL0020123-1,CL0020123-2, CL0020123-3, CL0020123-4, CL0020123-5,CL0020123-6, CL0020123-7, and CL0020123-8 32 HASQHINVWLSCDR-L1 for CL0020123 and variants CL0020123-1, CL0020123-2, CL0020123-3,CL0020123-4, CL0020123-5, CL0020123-6, CL0020123-7, and CL0020123-8 33KASNLHT CDR-L2 for CL0020123 and variants CL0020123-1,CL0020123-2, CL0020123-3, CL0020123-4, CL0020123-5,CL0020123-6, CL0020123-7, and CL0020123-8 34 QQGQTYPRTCDR-L3 for CL0020123 and variants CL0020123-1, CL0020123-2, CL0020123-3,CL0020123-4, CL0020123-5, CL0020123-6, CL0020123-7, and CL0020123-8 35QVQLVQSGAEVKKPGASVKVSCKASGFSIEDFYIHWVRQAPGQ CL0020123-1 V_(H)GLEWMGWIDPENGDSKYAPKFQGRATITADTSTSTAYMELSSLRSEDTAVYYCHADHGNYGSTMDYWGQGTLVTVSS 36DIQMTQSPSSLSASVGDRVTITCHASQHINVWLSWYQQKPGKAP CL0020123-1 VL;KLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ CL0020123-2 VL;QGQTYPRTFGQGTKVEIK CL0020123-3 VL; CL0020123-4 VL CL0020123-5 VL;CL0020123-6 VL; CL0020123-7 VL; CL0020123-8 VL 37QVQLVQSGAEVKKPGASVKVSCKASGFSIEDFYIHWVRQAPGQ CL0020123-2 V_(H)GLEWMGWIDPENGDSKYAPKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCHADHGNYGSTMDYWGQGTLVTVSS 38QVQLVQSGAEVKKPGASVKVSCKASGFSIEDFYIHWVRQAPGQ CL0020123-3 V_(H)GLEWMGWIDPEQGDSKYAPKFQGRATITADTSTSTAYMELSSLRSEDTAVYYCHADHGNYGSTMDYWGQGTLVTVSS 39 WIDPEQGDSKYAPKFQGCDR-H2 for variants CL0020123-3 and CL0020123-6 40QVQLVQSGAEVKKPGASVKVSCKASGFSIEDFYIHWVRQAPGQ CL0020123-4 V_(H)GLEWMGWIDPENGESKYAPKFQGRATITADTSTSTAYMELSSLRSEDTAVYYCHADHGNYGSTMDYWGQGTLVTVSS 41 WIDPENGESKYAPKFQGCDR-H2 for variants CL0020123-4 and CL0020123-7 42QVQLVQSGAEVKKPGASVKVSCKASGFSIEDFYIHWVRQAPGQ CL0020123-5 V_(H)GLEWMGWIDPEQGESKYAPKFQGRATITADTSTSTAYMELSSLRSEDTAVYYCHADHGNYGSTMDYWGQGTLVTVSS 43 WIDPEQGESKYAPKFQGCDR-H2 for variants CL0020123-5 and CL0020123-8 44QVQLVQSGAEVKKPGASVKVSCKASGFSIEDFYIHWVRQAPGQ CL0020123-6 V_(H)GLEWMGWIDPEQGDSKYAPKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCHADHGNYGSTMDYWGQGTLVTVSS 45QVQLVQSGAEVKKPGASVKVSCKASGFSIEDFYIHWVRQAPGQ CL0020123-7 V_(H)GLEWMGWIDPENGESKYAPKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCHADHGNYGSTMDYWGQGTLVTVSS 46QVQLVQSGAEVKKPGASVKVSCKASGFSIEDFYIHWVRQAPGQ CL0020123-8 V_(H)GLEWMGWIDPEQGESKYAPKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCHADHGNYGSTMDYWGQGTLVTVSS 47W-I-D-P-E-β₆-G-β₈-S-K-Y-A-P-K-F-Q-G, wherein β₆CDR-H2 consensus sequence is N or Q and β₈ is D or E 48G-F-T-F-T-α₆-F-Y-M-S, wherein α₆ is D or N CDR-H1 consensus sequence 49V-I-R-N-β₅-β₆-N-β₈-Y-T-β₁₁-β₁₂-Y-N-P-S-V-K-G, CDR-H2 consensus sequencewherein β₅ is K or R; β₆ is A or P; β₈ is G or A;β₁₁ is A or T; and β₁₂ is G or D 50γ₁-R-L-γ₄-Y-G-F-D-Y, wherein γ₁ is A or T; CDR-H3 consensus sequenceand γ₄ is T or S  51 Q-S-S-K-S-L-L-H-S-δ₁₀-G-K-T-Y-L-N, wherein δ₁₀CDR-L1 consensus sequence is N or T 52Q-Q-F-L-E-φ₆-P-F-T, wherein φ₆ is Y or F CDR-L3 consensus sequence 53AGGAATGTGGGGAGCACGGAG Primer sequence 54 TGCATCGCATTGTCTGAGTAGGTGPrimer sequence 55 ACCCTAGTCCTGACTGTTGCTC Primer sequence 56TATAGGAACTTCGCGACACGGACAC Primer sequence 57 ACAGGAGGGACCTACCTTCAGPrimer sequence 58 GCCTGCCTTTCAGAGACCTCAGTC Primer sequence 59CCTCTCCGGCTGCTCATCTTACTC Primer sequence 60 GTCTCTCAGCCCTGGCAGAGTTTGPrimer sequence 61 CGCCTACCCTAGTCCTGACTGTTG Primer sequence 62AAAGCCTACAGCATCCTCACCTC Primer sequence 63 GCATCATGGGGTTGTAGATTCCGPrimer sequence 64 GGGGS Linker sequence 65 HHHHHH 6X-His tag 66QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQSPG V_(H) for anti-TREM2 antibodyRGLEWIGRSDPTTGGTNYNEKFKTKATLTVDKPSSTAYMQLSSL RS9.F6TSDDSAVYYCVRTSGTGDYWGQGTSLTVSSAKTTAPSVYPLAP VCGGTTGSSVT 67DVVMTQTPLSLPVSLGDQASISCRSSQSLVFINNGNTFLHWYLQK VL for anti-TREM2 antibodyPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLG RS9.F6VYFCSQTTHVPPTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGA SVVCF 68DIVMTQSPDSLAVSLGERATINCQSSKSLLHSNGKTYLNWYQQK CL0020307-1 VLPGQPPKLLIYWMSTRASGVPDRFSGSGSGTDFTLTISSLQAEDVA VYYCQQFLEFPFTFGQGTKVEIK 69FPGESES TREM2 epitope 70 GEKGPCQRV TREM2 epitope 71 TLRNLQPHDAGLTREM2 epitope 72 VEVL TREM2 epitope 73DIQMTQSPSSVSASVGDRVTITCRASQGISNWLAWYQQKPGKAP Reference antibody #1 VLKLLIYAASSLQVGVPLRFSGSGSGTDFTLTISSLQPEDFATYYCQ QADSFPRNFGQGTKLEIK 74EVQLVQSGAEVKKPGESLKISCKGSGHSFTNYWIAWVRQMPGK Reference antibody #1 V_(H)GLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAVYFCARQRTFYYDSSGYFDYWGQGTLVTVSS 75DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAP Reference antibody #2 VLKLLIYAASSLQNGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQ ADSFPRTFGQGTKLEIK 76EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGK Reference antibody #2 V_(H)GLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYFCARQRTFYYDSSDYFDYWGQGTLVTVSS 77DVVMTQSPDSLAVSLGERATINCRSSQSLVHSNRYTYLHWYQQ Reference antibody #3 VLKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDV GVYYCSQSTRVPYTFGQGTKLEIK78 QVQLVQSGAEVKKPGASVKVSCKASGYAFSSQWMNWVRQAP Reference antibody #3 V_(H)GQRLEWIGRIYPGGGDTNYAGKFQGRVTITADTSASTAYMELSSLRSEDTAVYYCARLLRNQPGESYAMDYWGQGTLVTVSS 79EVQLVESGGGLVQPGGSLRLSCAGSGFTFTDFYMSWVRQAPGK CL0020188-4 VHGPEWLSVIRNKANGYTAGYNPSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLTYGFDYWGQGTLVTVSS 80 NGDS Table 5 hV_(H) 81 QGDSTable 5 hV_(H) 82 NGES Table 5 hV_(H) 83 QGES Table 5 hV_(H)

1. An isolated antibody or antigen-binding fragment thereof thatspecifically binds to a human TREM2, wherein the antibody orantigen-binding fragment thereof comprises: (a) a CDR-H1 sequencecomprising the sequence of GFSIEDFYIH (SEQ ID NO:29); (b) a CDR-H2sequence comprising the sequence of W-I-D-P-E-β₆-G-β₈-S—K-Y-A-P—K-F-Q-G(SEQ ID NO:47), wherein β₆ is N or Q and β₈ is D or E; (c) a CDR-H3sequence comprising the sequence of HADHGNYGSTMDY (SEQ ID NO:31); (d) aCDR-L1 sequence comprising the sequence of HASQHINVWLS (SEQ ID NO:32);(e) a CDR-L2 sequence comprising the sequence of KASNLHT (SEQ ID NO:33);and (f) a CDR-L3 sequence comprising the sequence of QQGQTYPRT (SEQ IDNO:34).
 2. The isolated antibody or antigen-binding fragment of claim 1,wherein the CDR-H2 sequence is selected from SEQ ID NOS:30, 39, 41, and43.
 3. The isolated antibody or antigen-binding fragment of claim 1,wherein the antibody or antigen-binding fragment comprises: (a) a CDR-H1comprising the amino acid sequence of SEQ ID NO:29, a CDR-H2 comprisingthe amino acid sequence of SEQ ID NO:30, a CDR-H3 comprising the aminoacid sequence of SEQ ID NO:31, a CDR-L1 comprising the amino acidsequence of SEQ ID NO:32, a CDR-L2 comprising the amino acid sequence ofSEQ ID NO:33, and a CDR-L3 comprising the amino acid sequence of SEQ IDNO:34; or (b) a CDR-H1 comprising the amino acid sequence of SEQ IDNO:29, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:39, aCDR-H3 comprising the amino acid sequence of SEQ ID NO:31, a CDR-L1comprising the amino acid sequence of SEQ ID NO:32, a CDR-L2 comprisingthe amino acid sequence of SEQ ID NO:33, and a CDR-L3 comprising theamino acid sequence of SEQ ID NO:34; or (c) a CDR-H1 comprising theamino acid sequence of SEQ ID NO:29, a CDR-H2 comprising the amino acidsequence of SEQ ID NO:41, a CDR-H3 comprising the amino acid sequence ofSEQ ID NO:31, a CDR-L1 comprising the amino acid sequence of SEQ IDNO:32, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:33, anda CDR-L3 comprising the amino acid sequence of SEQ ID NO:34; or (d) aCDR-H1 comprising the amino acid sequence of SEQ ID NO:29, a CDR-H2comprising the amino acid sequence of SEQ ID NO:43, a CDR-H3 comprisingthe amino acid sequence of SEQ ID NO:31, a CDR-L1 comprising the aminoacid sequence of SEQ ID NO:32, a CDR-L2 comprising the amino acidsequence of SEQ ID NO:33, and a CDR-L3 comprising the amino acidsequence of SEQ ID NO:34.
 4. The isolated antibody or antigen-bindingfragment of claim 1, comprising a V_(H) sequence that has at least 85%sequence identity to any one of SEQ ID NOS:27, 35, 37, 38, 40, 42, 44,45, and
 46. 5. The isolated antibody or antigen-binding fragment ofclaim 4, wherein the V_(H) sequence has at least 90% sequence identityto SEQ ID NO:27.
 6. The isolated antibody or antigen-binding fragment ofclaim 5, wherein the V_(H) sequence has at least 95% sequence identityto SEQ ID NO:27.
 7. The isolated antibody or antigen-binding fragment ofclaim 6, wherein the V_(H) sequence comprises SEQ ID NO:27.
 8. Theisolated antibody or antigen-binding fragment of claim 1, comprising aV_(L) sequence that has at least 85% sequence identity to SEQ ID NO:28or SEQ ID NO:36.
 9. The isolated antibody or antigen-binding fragment ofclaim 8, wherein the V_(L) sequence has at least 90% sequence identityto SEQ ID NO:28.
 10. The isolated antibody or antigen-binding fragmentof claim 9, wherein the V_(L) sequence has at least 95% sequenceidentity to SEQ ID NO:28.
 11. The isolated antibody or antigen-bindingfragment of claim 10, wherein the V_(L) sequence comprises SEQ ID NO:28.12. The isolated antibody or antigen binding fragment of claim 8,wherein the antibody or antigen-binding fragment comprises: (a) a V_(H)sequence comprising SEQ ID NO:27 and a V_(L) sequence comprising SEQ IDNO:28; or (b) a V_(H) sequence comprising SEQ ID NO:35 and a V_(L)sequence comprising SEQ ID NO:36; or (c) a V_(H) sequence comprising SEQID NO:37 and a V_(L) sequence comprising SEQ ID NO:36; or (d) a V_(H)sequence comprising SEQ ID NO:38 and a V_(L) sequence comprising SEQ IDNO:36; or (e) a V_(H) sequence comprising SEQ ID NO:40 and a V_(L)sequence comprising SEQ ID NO:36; or (f) a V_(H) sequence comprising SEQID NO:42 and a V_(L) sequence comprising SEQ ID NO:36; or (g) a V_(H)sequence comprising SEQ ID NO:44 and a V_(L) sequence comprising SEQ IDNO:36; or (h) a V_(H) sequence comprising SEQ ID NO:45 and a V_(L)sequence comprising SEQ ID NO:36; or (i) a V_(H) sequence comprising SEQID NO:46 and a V_(L) sequence comprising SEQ ID NO:36.
 13. The isolatedantibody or antigen-binding fragment thereof of claim 1, wherein theantigen-binding fragment is a Fab, a F(ab′)₂, a scFv, or a bivalentscFv.
 14. A pharmaceutical composition comprising the isolated antibodyor antigen-binding fragment thereof of claim 1 and a pharmaceuticallyacceptable carrier.
 15. An isolated polynucleotide comprising anucleotide sequence encoding the isolated antibody or antigen-bindingfragment thereof of claim
 1. 16. A method of treating aneurodegenerative disease in a subject, comprising administering to thesubject the isolated antibody or antigen-binding fragment thereof ofclaim
 1. 17. The method of claim 16, wherein the neurodegenerativedisease is selected from the group consisting of: Alzheimer's disease,primary age-related tauopathy, progressive supranuclear palsy (PSP),frontotemporal dementia, frontotemporal dementia with parkinsonismlinked to chromosome 17, argyrophilic grain dementia, amyotrophiclateral sclerosis, amyotrophic lateral sclerosis/parkinsonism-dementiacomplex of Guam (ALS-PDC), corticobasal degeneration, chronic traumaticencephalopathy, Creutzfeldt-Jakob disease, dementia pugilistica, diffuseneurofibrillary tangles with calcification, Down's syndrome, familialBritish dementia, familial Danish dementia,Gerstmann-Straussler-Scheinker disease, globular glial tauopathy,Guadeloupean parkinsonism with dementia, Guadelopean PSP,Hallevorden-Spatz disease, hereditary diffuse leukoencephalopathy withspheroids (HDLS), Huntington's disease, inclusion-body myositis,multiple system atrophy, myotonic dystrophy, Nasu-Hakola disease,neurofibrillary tangle-predominant dementia, Niemann-Pick disease typeC, pallido-ponto-nigral degeneration, Parkinson's disease, Pick'sdisease, postencephalitic parkinsonism, prion protein cerebral amyloidangiopathy, progressive subcortical gliosis, subacute sclerosingpanencephalitis, and tangle only dementia.
 18. A method of decreasinglevels of sTREM2 in a subject having a neurodegenerative disease,comprising administering to the subject the isolated antibody orantigen-binding fragment thereof of claim
 1. 19. A method of enhancingTREM2 activity in a subject having a neurodegenerative disease,comprising administering to the subject the isolated antibody orantigen-binding fragment thereof of claim 1.